Methods for efficient, expansive, user-defined DNA mutagenesis

ABSTRACT

Methods for the creation of one or more user-defined mutations that can be located anywhere in a target sequence, such as in a gene are disclosed. These methods can be used on a single-stranded or double-stranded template and, for a single-stranded template, generally include: (a) conducting a first amplification reaction in the presence of a thermostable DNA polymerase and a thermostable DNA ligase to synthesize a mutagenized strand of DNA comprising at least one mutagenic oligonucleotide relative to a complementary single-stranded template DNA comprising a target nucleic acid molecule in a circular DNA vector; (b) conducting a second amplification reaction in the presence of a thermostable DNA polymerase and a thermostable DNA ligase to synthesize a complementary mutant strand of DNA; and (c) degrading the template DNA and non-covalently closed circular nucleic acid molecules to obtain a mutation-containing double-stranded DNA product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. National Stage applicationNo. 14/407,683 filed on Dec. 12, 2014, now U.S. Pat. No. 9,458,453issued on Oct. 4, 2016, which is a 35 U.S.C. §371 U.S. national phaseentry of International Application No. PCT/US13/45487 having aninternational filing date of Jun. 12, 2013, which claims the benefit ofU.S. Provisional Application No. 61/658,607, filed Jun. 12, 2012, whichare incorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DEB-0950939awarded by the National Science Foundation (NSF). The government hascertain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“111232-00544_ST25.txt”. The sequence listing is 8,192 bytes in size,and was created on Aug. 25, 2016. It is hereby incorporated by referencein its entirety.

BACKGROUND

An efficient and high-throughput mutagenesis strategy is an integralpart of protein structure/function studies, directed evolutionexperiments for the discovery of novel proteins, and optimization ofgenetic elements in synthetic biology systems. Among the methods for invitro mutagenesis known in the art, none offers a convenient, efficientand high-throughput approach for creating an extensive, user-definedlibrary of variants in which single or multiple mutations can be locatedat any position. For example, site-directed mutagenesis methods, such asKunkel mutagenesis (Kunkel, 1985), QuikChange (QuikChange Site-DirectedMutagenesis Kit, Stratagene), and inverse PCR (Dominy and Andrews,2003), are low-throughput methods. Combined chain reaction requiresspecially designed sets of primers and cloning of PCR products (Hames etal., 2005; Bi and Stambrook, 1998). Creating mutations by gene synthesisis comparatively expensive and requires sub-cloning of DNA. Error-pronePCR suffers from mutational bias, the inability to define the mutationalcomposition, and the inability to effectively cause most amino acidsubstitutions, which require two or three mutations in a single codon.Methods that rely on random DNA cleavage reagents or transposons formutating short sequences of DNA suffer from complex procedures and theinability to target the mutations (Baldwin et al., 2008; Murakami etal., 2002; Liu and Cropp, 2012).

Kunkel mutagenesis is a site-directed method developed to introducemutations by using a mutation-encoding oligonucleotide (oligo) thatanneals to a single-stranded uracil-containing circular DNA template. T7DNA polymerase and T4 ligase are used to complete synthesis of themutated strand. Upon transformation of E. coli, the newly synthesizedmutated strand survives to a higher extent than the uracil-containingtemplate strand. While the initial Kunkel protocol described makingsingle base substitutions (Kunkel, 1987), other researchers have adaptedthe method for creating site-saturation libraries in a single codon(Scholle et al., 2005; Weiss et al., 2000). The mutational efficiency ofsite-directed Kunkel mutagenesis is limited such that typically 50-70%of transformed colonies harbor the desired mutation, while the remainderharbor the wildtype sequence (Kunkel et al., 1987).

Existing methods for site-directed mutagenesis at multiple distal sitessimultaneously either have complex and multi-step procedures or have notbeen demonstrated to be efficient enough for library construction (Biand Stambrook, 1998; QuikChange Multi Site-Directed Mutagenesis Kit,Stratagene). In addition, the mutagenesis toolbox currently lacks amethod for creating extensive DNA libraries with a researcher-definedmutational composition spanning across an entire gene. For example,until now there has been no efficient method to make a librarycomprising all 18,900 possible single codon substitutions of a 300 aminoacid long protein, nor is there an efficient method to make auser-prescribed subset of only 2000 of these 18,900 mutations.Accordingly, current methods to make multiple mutations simultaneouslysuffer from complicated procedures or low efficiencies and methods forsite-directed mutagenesis for creating single mutations suffer fromsub-optimal efficiency and variable success.

SUMMARY

In some aspects, the presently disclosed subject matter provides methodsfor creating one or more user-defined mutations that can be locatedanywhere in a target sequence, such as in a gene. These mutations cancomprise single mutations, multiple mutations, or a comprehensive codonmutagenesis library, in which all possible single codon substitutions ina gene may be created.

In other aspects, the presently disclosed subject matter provides amethod for introducing one or more mutations to a single-stranded targetnucleic acid molecule, the method comprising: (a) providing asingle-stranded uracil-containing template comprising a target nucleicacid molecule in a circular DNA vector; (b) annealing at least onemutagenic oligonucleotide comprising at least one mutation to the targetnucleic acid molecule at a first elevated temperature; (c) conducting afirst amplification reaction in the presence of a thermostable DNApolymerase and a thermostable DNA ligase at a second elevatedtemperature to synthesize a mutagenized strand of DNA comprising the atleast one mutagenic oligonucleotide; (d) denaturing the mutagenizedstrand of DNA at a third elevated temperature; (e) annealing a reverseprimer to the mutagenized strand of DNA at a fourth elevatedtemperature; (f) conducting a second amplification reaction in thepresence of a thermostable DNA polymerase and a thermostable DNA ligaseat a fifth elevated temperature to synthesize a complementary mutantstrand of DNA; and (g) degrading the uracil-containing DNA andnon-covalently closed circular nucleic acid molecules at a sixthelevated temperature to obtain a mutation-containing double-stranded DNAproduct.

In further aspects, the presently disclosed subject matter provides amethod for introducing one or more mutations to a double-stranded targetnucleic acid molecule, the method comprising: (a) providing adouble-stranded uracil-containing template comprising a target nucleicacid molecule in a circular DNA vector; (b) denaturing the targetnucleic acid molecule at a first elevated temperature; (c) annealing atleast one mutagenesis oligonucleotide comprising at least one mutationto the target nucleic acid molecule at a second elevated temperature;(d) conducting a first amplification reaction in the presence of athermostable DNA polymerase and a thermostable DNA ligase at a thirdelevated temperature to synthesize a mutagenized strand of DNAcomprising the at least one mutagenesis oligonucleotide; (e) degradingthe uracil-containing DNA and non-covalently closed circular nucleicacid molecules at a fourth elevated temperature; (f) denaturing themutagenized strand of DNA at a fifth elevated temperature; (g) annealinga reverse primer to the mutagenized strand of DNA at a sixth elevatedtemperature; and (h) conducting a second amplification reaction in thepresence of a thermostable DNA polymerase and a thermostable DNA ligaseat a seventh elevated temperature to synthesize a complementary mutantstrand of DNA to obtain a mutation-containing double-stranded DNAproduct.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows a schematic diagram of PFunkel mutagenesis using a ssDNAtemplate. The basic protocol is depicted. For multiple-site mutagenesis,the addition of the polymerase, dNTPs, ligase, DTT, and NAD+ may bedelayed until after the first annealing step. For comprehensive codonmutagenesis, the ratio of oligonucleotide to template is kept low tominimize multiple mutations in a single reaction product. Cycling withoccasional spiking of additional mutagenic oligonucleotides improves thereaction yield;

FIG. 2 shows various stages and control reactions for PFunkelsite-directed mutagenesis. DNA was visualized on a 1% agarose gel runfor 60 min at 75 V. Lane 1: 1 kB ladder. Lane 2: uracil-containing ssDNAtemplate prep. Lane 3: pSkunk3-BLA phagemid mini-prep. Lanes 4-6:PFunkel reaction terminated after mutant second strand ligation step.Lane 4: no oligonucleotides added. Lane 5: no ligase added. Lane 6:oligonucleotides and ligase added. Lane 7: PFunkel reaction terminatedafter third mutagenic strand ligation step. The white triangle indicatesthe appearance of a band at the correct size to be phagemid cccDNA. Lane8: The complete reaction prior to transformation;

FIG. 3 shows a schematic diagram of the Matlab algorithm for designingthe mutagenic oligonucleotides for comprehensive codon mutagenesis;

FIG. 4A and FIG. 4B show completeness and frequency of codonsubstitutions observed in 454 sequencing of the comprehensive codonmutagenesis library of TEM-1: (a) Number of the 63 possible codonsubstitutions observed and (b) frequency of codon substitutions observedas a function of position in the gene;

FIG. 5A and FIG. 5B show the distribution of the frequency of the typeof (a) codon substitutions and (b) codons substituted into thecomprehensive codon mutagenesis library CCM-1;

FIG. 6 shows tazobactam resistance of selected alleles. The increase inampicillin or piperacillin resistance is reported as the fold increase(over TEM-1) in the minimum inhibitory concentration (MIC) of theantibiotic in the presence of 6 μg/mL tazobactam. MIC assays performedin √2-fold increments of antibiotic concentration. Median MIC values ofthree replicates were used; and

FIG. 7 shows a schematic diagram of PFunkel using a dsDNA template.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

I. PFUNKEL MUTAGENESIS

A. Overview

The presently disclosed subject matter provides versatile and efficientmethods for creating user-defined mutations that can be located anywherein a target sequence. The presently disclosed methods allow for thecreation of gene libraries or individual constructs where each memberreceives one or multiple user-defined mutations at any position.

In some embodiments, the presently disclosed methods allow for a newtype of mutagenesis library, a comprehensive codon mutagenesis library,in which all possible single codon substitutions in a gene are created(also termed comprehensive site-saturation mutagenesis). A comprehensivecodon mutagenesis library comprises every possible codon substitution inthe gene with the goal of only one codon substitution per library member(i.e., library members containing more than one codon mutated aredesired to be minimized). Such a library is the equivalent of creating asite-saturation mutagenesis library at all positions in the gene. Inother embodiments, the presently disclosed methods can be used to makenon-comprehensive libraries, in which not every possible single codonsubstitution in a gene is created. In some other embodiments, thepresently disclosed methods can be used to make non-comprehensive, butextensive libraries, where some, many or most of the possible singlecodon substitutions in a gene are created.

A significant limitation of Kunkel mutagenesis is the high frequency ofwild-type sequences (10-50%) (Kunkel et al., 1987). Without wishing tobe bound to any one particular theory, it is postulated that wild-typesequences arise for two reasons. First, the low operating temperature ofthe second strand synthesis step allows “junk” DNA present in the singlestranded DNA prep to prime the single stranded DNA. Such synthesis caneither create a wild-type double stranded product or “poison” amutation-bearing product by creating reaction side-products that possessa nick or a displaced strand (Wassman et al., 2004). Additionally, atlower temperatures, the mutagenic oligonucleotides are more prone toanneal non-specifically to the template. Such reaction side-productsthat are not in the covalently closed circular DNA (cccDNA) form areprone to degradation by cellular nucleases, removing the mutation. Thepresence of such junk DNA is apparent from DNA gels of reaction productsin which no mutagenic oligonucleotides were added, yet higher molecularweight products are produced (Kunkel et al., 1991). The secondpostulated reason for the high occurrence of wild-type sequences inKunkel mutagenesis is the repair of the mutation by mismatch-repairmachinery or repair of the uracil-containing template strand in the cellafter transformation.

Although there are some similarities between Kunkel mutagenesis and thepresently disclosed methods (termed PFunkel mutagenesis), there are anumber of key differences that serve to increase the efficiency of thereaction and minimize the appearance of wildtype sequences in theresulting library. At least some of the major differences include, butare not limited to: (a) the use of a thermostable DNA polymerase andligase, which enables a shift in the operating temperature of thereaction from 25° C.-37° C. to 55° C.-95° C.; (b) the option to usePCR-like thermal cycling and stepwise addition of oligonucleotides totailor the average number of mutations per gene; (c) synthesis of asecond mutated strand complementary to the first mutated strand thatdisplaces the template strand; and (d) in vitro degradation of theuracil-containing template and DNA products not in the desired cccDNAform by the addition of uracil DNA glycosylase (UDG) and an exonuclease,such as exonuclease III (Exo III). Additionally, in some embodiments,PFunkel mutagenesis can be performed on any dsDNA plasmid template andavoids the use of phage.

The in vitro reaction steps of PFunkel mutagenesis (FIG. 1, FIG. 7,Table 4) are designed to eliminate products other than the desiredmutated cccDNA plasmid molecules, resulting in high mutationalefficiencies. In some embodiments, other than the initial kinasereaction to phosphorylate the mutagenic oligonucleotides, all reactionsteps are conveniently performed in the same tube. In other embodiments,no DNA purification is required, except as an optional final step toimprove transformation efficiency. In still other embodiments, PFunkelmutagenesis is conveniently performed in a thermocycler.

In some embodiments, the target sequence is a gene. In otherembodiments, single mutations can be made using the presently disclosedmethods that approach 100% efficiency. In addition, multiple mutationsin a target sequence are made at high efficiency. It has been found thatincreasing the molar ratio of mutagenic oligonucleotide touracil-containing template increases multiple mutations and decreasingthe molar ratio makes only one mutation per molecule more likely. Inthis way, the number of mutations per molecule can be controlled.

In some embodiments, expansive, user-defined libraries of mutations arecreated, which comprise some, most, or all possible mutations of a geneor genes or a user-defined subset of all mutations of a gene or genes.As an example of a user-defined subset, comprehensive alanine scanningmutagenesis can be performed using the presently disclosed methods. Theability to make a library comprising all possible mutations of a geneallows the determination of the contribution of each of the residues ina particular protein, for example, to the stability and/or function ofthe protein. In other embodiments, some, most, or each nucleotide of atarget nucleic acid molecule can be substituted to determine the effecton the function of the target nucleic acid molecule. For example, targetnucleic acid molecules comprising genes, promoters, enhancers,silencers, insulators, activators, repressors, and the like can beexamined by creating a non-comprehensive or comprehensive library ofmutations according to the presently disclosed subject matter.Accordingly, in some embodiments, a non-comprehensive or comprehensivecodon mutagenesis library is used to obtain an improved protein, gene orgene promoter.

The presently disclosed subject matter can be used to make DNA mutationsor DNA mutation libraries with any desired target sequence, such as agene or genes. The presently disclosed methods also allow the creationof mutations that comprise the deletion or insertion of one or more DNAbases, instead of substitution mutations. The presently disclosedmethods allow for introducing one or multiple mutations per variant.

In some embodiments, for making mutations at multiple sites at distantsites in a target sequence simultaneously, the basic PFunkel mutagenesisprotocol is modified to increase the frequency of multiple mutations. Insome embodiments, the polymerase is added only after the annealing ofthe mutagenic oligonucleotides. Without wishing to be bound to any oneparticular theory, the rationale for the delayed addition of polymeraseis to prevent a bias for mutations that result from oligonucleotidesthat anneal efficiently. DNA synthesis from such early annealingoligonucleotides might proceed to regions of the gene where otheroligonucleotides are intended to anneal before the oligonucleotides forthose locations have a chance to anneal, thus decreasing the frequencyof multiple mutations in the resulting transformants. In otherembodiments, the extension temperature is reduced from 68° C. to 65° C.,to better ensure that the DNA polymerase, such as PfuTurbo Cx, does notstrand displace. Strand displacement of a strand created from onemutagenic oligonucleotide by a strand being synthesized starting from asecond mutagenic oligonucleotide would also reduce the frequency ofmultiple mutations.

PFunkel mutagenesis was designed in part to allow efficient constructionof libraries in which site-saturation mutagenesis (or any user-definedmutational composition) can be performed at multiple sitessimultaneously in a single reaction.

B. Mutagenesis Using a Single-Stranded Template

In some embodiments, the presently disclosed subject matter provides amethod for introducing one or more mutations to a single-stranded targetnucleic acid molecule, the method comprising: (a) providing asingle-stranded uracil-containing template comprising a target nucleicacid molecule in a circular DNA vector; (b) annealing at least onemutagenic oligonucleotide comprising at least one mutation to the targetnucleic acid molecule at a first elevated temperature; (c) conducting afirst amplification reaction in the presence of a thermostable DNApolymerase and a thermostable DNA ligase at a second elevatedtemperature to synthesize a mutagenized strand of DNA comprising the atleast one mutagenic oligonucleotide; (d) denaturing the mutagenizedstrand of DNA at a third elevated temperature; (e) annealing a reverseprimer to the mutagenized strand of DNA at a fourth elevatedtemperature; (f) conducting a second amplification reaction in thepresence of a thermostable DNA polymerase and a thermostable DNA ligaseat a fifth elevated temperature to synthesize a complementary mutantstrand of DNA; and (g) degrading the uracil-containing DNA andnon-covalently closed circular nucleic acid molecules at a sixthelevated temperature to obtain a mutation-containing double-stranded DNAproduct.

In some embodiments, the first elevated temperature is from about 50° C.to about 60° C. In other embodiments, the second elevated temperature isfrom about 60° C. to about 73° C. In still other embodiments, the thirdelevated temperature is from about 90° C. to about 98° C. In furtherembodiments, the fourth elevated temperature is about 50° C. to about60° C. In still further embodiments, the fifth elevated temperature isfrom about 60° C. to about 73° C. In some embodiments, the sixthelevated temperature is from about 32° C. to about 42° C. In otherembodiments, the second elevated temperature and the fifth elevatedtemperature are about 65° C.

In some embodiments, the presently disclosed methods comprise providinga single-stranded uracil-containing template. In other embodiments, auracil-containing single-stranded DNA (ssDNA) template containing thegene to be mutated is prepared. In some embodiments, the presentlydisclosed method comprises producing uracil-containing ssDNA bypropagating phagemid DNA comprising the DNA to be mutated in an E. colidut-1 ung-1 host, then infecting the culture with m13 helper phage andharvesting ssDNA from the resulting phage particles. E. coli dut-1 ung-1strains express a heat-sensitive dUTPase that has 5% of wildtypeactivity at 25° C. but <1% at 37° C., and are deficient in uracil DNAglycosylase activity (Taylor and Weiss, 1982). The result of thesemutations is the accumulation of high levels of intracellular dUTP thatbecomes incorporated in DNA in place of dTTP during DNA synthesis and isnot removed due to the lack of UDG activity. In other embodiments, ssDNApreparation takes only a day and requires no special laboratoryequipment or highly specialized training (Kunkel et al., 1991). In stillother embodiments, the template is prepared by using an m13 helper phageand CJ236, an E. coli strain that allows for uracil incorporation intoDNA.

In some embodiments, the presently disclosed methods comprise designingmutagenic oligonucleotides. In other embodiments, the mutagenicoligonucleotides are computationally designed. In some otherembodiments, the mutagenic oligonucleotide sequences are optimized byusing multiple parameters including length, melting temperature, GCcontent, hairpin and dimer formation, polynucleotide repeats, andhybridization energy. In further embodiments, a single mix of thedesired oligonucleotides is synthesized in a high-throughput chip formatthat allows for up to 3918 different oligonucleotides to be synthesizedin parallel. In some embodiments, a mutagenic oligonucleotide encoding amutation is first 5′ phosphorylated in a kinase reaction.

In some embodiments, the target nucleic acid molecule is denaturedbefore annealing at least one mutagenic oligonucleotide to the targetnucleic acid molecule. In other embodiments, the target nucleic acidmolecule is denatured at a temperature from about 90° C. to about 98° C.In still other embodiments, the target nucleic acid molecule is notdenatured before annealing at least one mutagenic oligonucleotide to thetarget nucleic acid molecule. Without wishing to be bound to any oneparticular theory, it is though that when high-temperature denaturationof the single-stranded DNA does not occur, the error rate is lowered.

In the annealing step, the oligonucleotides are mixed with thesingle-stranded template DNA in the presence of a DNA polymerase, DNAligase and dNTPs. In some embodiments, the DNA polymerase is athermostable DNA polymerase. In other embodiments, the thermostable DNApolymerase is selected from the group consisting of PfuTurbo Cx DNApolymerase, Taq DNA polymerase, Pfu DNA polymerase, PfuTurbo DNApolymerase, Tma DNA polymerase, Tli DNA polymerase, KOD DNA polymerase,JDF-3 DNA polymerase, PGB-D DNA polymerase, Tgo DNA polymerase, andPyrolobus furmarius DNA polymerase. In a preferred embodiment, thethermostable DNA polymerase is PfuTurbo Cx. In still other embodiments,the DNA ligase is a thermostable DNA ligase. In further embodiments, thethermostable DNA ligase is selected from the group consisting of Pfu DNAligase, Tth DNA ligase, Taq DNA ligase, Thermus filiformis ligase,Rhodothermus marinus DNA ligase, Thermus scotoductus DNA ligase andBacillus stearothermophilus DNA ligase.

In some embodiments, the annealing step and the synthesizing step occursat the same time. Therefore, in some embodiments, step (b) and step (c)are performed at the same time. In other embodiments, step (e) and step(f) are performed at the same time.

In some embodiments, the annealing and synthesizing step occurs morethan once to produce even more mutation-containing double-stranded DNAproduct. Therefore, in further embodiments, step (b) and step (c) areperformed more than once before step (d) occurs. In still furtherembodiments, when step (b) and step (c) are performed more than once, atleast one additional mutagenic oligonucleotide is added to the reactionin a stepwise manner. In other words, in some embodiments, theincubation temperatures are cycled to perform a denaturing, annealing,extension, and ligation step to complete the mutated second strand andseal the nick. In further embodiments, a second primer that anneals tothe new strand outside the gene is added to the reaction, and thedenaturing, annealing, extension, and ligation steps are repeated.

In some embodiments, a further step after step (c) and before step (d)comprises completing the ligation at a temperature from about 40° C. toabout 50° C. In other embodiments, a further step after step (f) andbefore step (g) comprises completing the ligation at a temperature fromabout 40° C. to about 50° C.

In some embodiments, the presently disclosed methods further comprisetransforming a host cell with the DNA product from step (g). In otherembodiments, the DNA product is purified before being transformed intothe host cell. In still other embodiments, the product is transformedinto an E. coli strain deficient in mismatch repair, which allows theuracil template strand to be degraded and replaced with a new strandcontaining the desired mutations. In further embodiments, uracil DNAglycosylase and exonuclease III are used to degrade theuracil-containing DNA and non-covalently closed circular nucleic acidmolecules. In further embodiments, the E. coli strain used is ES1301.

In some embodiments, the mutation-containing double-stranded DNA productcomprises one mutation. In other embodiments, when themutation-containing double-stranded DNA product comprises one mutation,the efficiency of mutagenesis is more than about 70%, such as more thanabout 75%, more than about 80%, more than about 85%, more than about90%, more than about 95%, more than about 96%, more than about 97%, morethan about 98%, more than about 99%, or about 100%. In some otherembodiments, when one mutation is made in the target nucleic acidmolecule, the efficiency of mutagenesis is more than about 98%. Infurther embodiments, when the mutation-containing double-stranded DNAproduct comprises one mutation, a 4:1 molar ratio of mutagenicoligonucleotide to single-stranded uracil-containing template is used.In still further embodiments, the ratio of mutagenic oligonucleotide tosingle-stranded uracil-containing template is from about 1:1 to about100:1.

In some embodiments, the mutation-containing double-stranded DNA productcomprises more than one mutation. In other embodiments, when themutation-containing double-stranded DNA product comprises more than onemutation, the efficiency of mutatgenesis is more than about 60%, such asmore than about 70%, more than about 75%, more than about 80%, more thanabout 85%, more than about 90%, more than about 95%, more than about96%, more than about 97%, more than about 98%, more than about 99%, orabout 100%. In some other embodiments, when more than one mutation ismade in the target nucleic acid molecule, the efficiency of mutagenesisis more than about 60%. In some other embodiments, when themutation-containing double-stranded DNA product comprises more than onemutation, a 1:20 molar ratio of mutagenic oligonucleotide tosingle-stranded uracil-containing template is used. In furtherembodiments, the ratio of mutagenic oligonucleotide to single-strandeduracil-containing template is from about 1:1 to about 100:1.

In some embodiments, the mutation-containing double-stranded DNA productcomprises a gene. In other embodiments, a non-comprehensive orcomprehensive codon mutagenesis library is created.

In some embodiments, at least one mutation is a substitution mutation, adeletion, or an addition of a DNA base or DNA bases.

In some embodiments, to minimize second strand synthesis originatingfrom junk ssDNA annealing to the uracil-containing ssDNA template, theoperating temperature is shifted from 25° C.-37° C. (as in the Kunkelmethod) to 55° C.-95° C. (FIG. 1). This operating temperature requires ahigh-fidelity thermostable polymerase capable of using auracil-containing template. Additionally, in some embodiments, apolymerase lacking strand displacement activity is used for creatingmultiple mutations simultaneously at different sites in a gene. Anexample of a commercially available polymerase that meets these criteriais PfuTurbo Cx (Agilent Technologies, Santa Clara, Calif., USA), avariant of Pfu polymerase with a V93Q mutation (Nørholm, 2010). Thismutation inactivates the uracil-binding pocket of the enzyme that wouldnormally cause it to stall at uracil bases. At less than or equal to 68°C., PfuTurbo Cx does not strand-displace, but still maintainssignificant polymerase activity (Hogrefe et al., 2001).

After the mutant strand is synthesized, the DNA nick is closed using aligase. In some embodiments, Taq ligase is chosen due to itseffectiveness in ligating DNA nicks, robust activity from 45° C. to 65°C., and ability to withstand many rounds of temperature cycling.

To minimize mismatch repair after transformation, reaction steps weredesigned to create the desired mutation-containing dsDNA product anddegrade non-cccDNA side-products and the uracil-containing template. Tocreate the dsDNA with the designed mutation on both strands, an excessof a ‘reverse’ oligonucleotide that anneals outside of the gene on thenewly created mutant strand can be added, such that it primes synthesisof a new complementary strand that encodes the desired mutations anddisplaces the uracil-containing template. Treatment with UDG acts toexcise the uracil bases from the original template strands leavingapyrimidinic (AP) sites. Treatment with an exonuclease, such asexonuclease III (ExoIII), which has both AP-site endonuclease and 3′->5′exonuclease activity (Rogers and Weiss, 1980), acts to create nicks atthe AP sites and then digests the template strand at the nicks and fromany 3′ end in the context of dsDNA. Therefore, in some embodiments, ExoIII and UDG are added to the reaction to remove the template andundesired side-products. In other embodiments, all the steps for thisprocedure take about three hours to complete.

In some embodiments, the DNA is transformed into E. coli without beingpurified first. In other embodiments, the DNA is purified before beingtransformed into E. coli.

In other embodiments, the mutations are introduced via oligonucleotidesthat anneal and prime synthesis on a single-stranded uracil-containingDNA template. In other embodiments, the reaction makes use of fiveenzymes and undergoes multiple temperature cycles that ultimately resultin double-stranded closed circular plasmid DNA ready for transformation,each molecule encoding a user-defined mutation.

In further embodiments, the presently disclosed subject matter providesa method for introducing one or more mutations to a single-strandedtarget nucleic acid molecule, the method comprising: (a) providing asingle-stranded uracil-containing template comprising a target nucleicacid molecule in a circular DNA vector; (b) annealing at least onemutagenic oligonucleotide comprising at least one mutation to the targetnucleic acid molecule at a first elevated temperature; (c) conducting afirst amplification reaction in the presence of a thermostable DNApolymerase and a thermostable DNA ligase at a second elevatedtemperature to synthesize a mutagenized strand of DNA comprising the atleast one mutagenic oligonucleotide; (d) denaturing the mutagenizedstrand of DNA at a third elevated temperature; (e) annealing a reverseprimer to the mutagenized strand of DNA at a fourth elevatedtemperature; and (f) conducting a second amplification reaction in thepresence of a thermostable DNA polymerase and a thermostable DNA ligaseat a fifth elevated temperature to synthesize a complementary mutantstrand of DNA; (g) degrading the uracil-containing DNA by transformingthe products of the second amplification reaction into E. coli.

The presently disclosed methods can be optimized for introducing one ormore mutations per gene and the frequency of mutated variants canapproach 100%. Importantly, the method is rapid, taking a single day andcan be performed in a single tube.

C. Mutagenesis Using a Double-Stranded Template (Phage-Less PFunkelMutagenesis)

The presently disclosed subject methods also can be performed byremoving the phage-assisted single-stranded DNA preparation step, tomake the method as widely accessible as possible. The methods vary fromthe methods utilizing a single-stranded template for some of the steps,for example, but the principles of the methods are similar to theprinciples described hereinabove for a single-stranded template.

This protocol does not require bacteriophage and therefore avoids theneed to move the target nucleic acid template from a normal plasmidvector to an appropriate phagemid. In some embodiments, the CJ236 E.coli strain is used to prepare the uracil-containing template DNA. Inother embodiments, a standard mini-prep procedure is used to obtain theDNA from the E. coli.

The protocol used is similar to the PFunkel mutagenesis protocoldescribed hereinabove except for a few minor changes. For example, thedU-single-stranded DNA template is replaced with a dU-double-strandedDNA template. Also, the order of steps is changed such that digestion ofthe template is performed directly after the first mutant strand issynthesized and ligated. In some embodiments, exonuclease I is addedalong with exoIII and UDG to improve degradation of the template. Thereverse primer step is then performed directly afterwards.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for introducing one or more mutations to adouble-stranded target nucleic acid molecule, the method comprising: (a)providing a double-stranded uracil-containing template comprising atarget nucleic acid molecule in a circular DNA vector; (b) denaturingthe target nucleic acid molecule at a first elevated temperature; (c)annealing at least one mutagenesis oligonucleotide comprising at leastone mutation to the target nucleic acid molecule at a second elevatedtemperature; (d) conducting a first amplification reaction in thepresence of a thermostable DNA polymerase and a thermostable DNA ligaseat a third elevated temperature to synthesize a mutagenized strand ofDNA comprising the at least one mutagenesis oligonucleotide; (e)degrading the uracil-containing DNA and non-covalently closed circularnucleic acid molecules at a fourth elevated temperature; (f) denaturingthe mutagenized strand of DNA at a fifth elevated temperature; (g)annealing a reverse primer to the mutagenized strand of DNA at a sixthelevated temperature; and (h) conducting a second amplification reactionin the presence of a thermostable DNA polymerase and a thermostable DNAligase at a seventh elevated temperature to synthesize a complementarymutant strand of DNA to obtain a mutation-containing double-stranded DNAproduct.

In some embodiments, the first elevated temperature is from about 90° C.to about 98° C. In other embodiments, the second elevated temperature isfrom about 50° C. to about 60° C. In still other embodiments, the thirdelevated temperature is from about 60° C. to about 73° C. In furtherembodiments, the fourth elevated temperature is from about 32° C. toabout 42° C. In still further embodiments, the fifth elevatedtemperature is from about 90° C. to about 98° C. In some embodiments,the sixth elevated temperature is from about 50° C. to about 60° C. Inother embodiments, the seventh elevated temperature is from about 60° C.to about 73° C. In some embodiments, the third elevated temperature andthe seventh elevated temperature are about 65° C.

The annealing step comprises the use of a DNA polymerase and a DNAligase. In some embodiments, the DNA polymerase is a thermostable DNApolymerase. In other embodiments, the thermostable DNA polymerase isselected from the group consisting of PfuTurbo Cx DNA polymerase, TaqDNA polymerase, Pfu DNA polymerase, PfuTurbo DNA polymerase, Tma DNApolymerase, Tli DNA polymerase, KOD DNA polymerase, JDF-3 DNApolymerase, PGB-D DNA polymerase, Tgo DNA polymerase and Pyrolobusfurmarius DNA polymerase. In a preferred embodiment, the thermostableDNA polymerase is PfuTurbo Cx. In some other embodiments, the DNA ligaseis a thermostable DNA ligase. In further embodiments, the thermostableDNA ligase is selected from the group consisting of Pfu DNA ligase, TthDNA ligase, Taq DNA ligase, Thermus filiformis ligase, Rhodothermusmarinus DNA ligase, Thermus scotoductus DNA ligase and Bacillusstearothermophilus DNA ligase.

In some embodiments, step (c) and step (d) are performed at the sametime. In other embodiments, step (g) and step (h) are performed at thesame time.

In some embodiments, step (c) and step (d) are performed more than oncebefore step (e) occurs. In other embodiments, when step (c) and step (d)are performed more than once, at least one additional mutagenicoligonucleotide is added to the reaction in a stepwise manner.

In further embodiments, uracil DNA glycosylase and exonuclease III areused to degrade the uracil-containing DNA and non-covalently closedcircular nucleic acid molecules after the first amplification reactionoccurs.

In some embodiments, a further step after step (d) and before step (e)comprises completing the ligation at a temperature from about 40° C. toabout 50° C. In other embodiments, a further step after step (h)comprises completing the ligation at a temperature from about 40° C. toabout 50° C.

In some embodiments, the mutation-containing double-stranded DNA productcomprises one mutation. In other embodiments, the efficiency ofmutagenesis is more than about 98%. In still other embodiments, a 4:1molar ratio of mutagenic oligonucleotide to double-strandeduracil-containing template is used. In further embodiments, the ratio ofmutagenic oligonucleotide to double-stranded uracil-containing templateis from about 1:1 to about 100:1.

In some embodiments, the mutation-containing double-stranded DNA productcomprises more than one mutation.

In some embodiments, the mutation-containing double-stranded DNA productcomprises a gene. In other embodiments, a non-comprehensive orcomprehensive codon mutagenesis library is created.

In some embodiments, at least one mutation is a substitution mutation, adeletion, or an addition of a DNA base.

In some embodiments, the presently disclosed methods further comprisetransforming a host cell with the DNA product from step (h). In otherembodiments, the DNA product is purified before being transformed intothe host cell.

D. Comprehensive or Non-Comprehensive Codon Mutagenesis Libraries

The presently disclosed methods allow the creation of a new type ofmutagenesis library, a comprehensive codon mutagenesis library, in whichall possible single codon substitutions in a gene are created (this canalso be called comprehensive site-saturation mutagenesis). Acomprehensive codon mutagenesis library comprises every possible codonsubstitution in the gene with only one codon substitution per librarymember (i.e., library members containing more than one codon mutated aredesired to be minimized). Such a library is the equivalent of creating asite-saturation mutagenesis library at all positions in the gene.

The presently disclosed methods also allow the creation of anon-comprehensive codon mutagenesis library. By “non-comprehensive”, itis meant that the mutation-containing double-stranded DNA products fromthe presently disclosed methods do not encompass all possiblemutation-containing double-stranded DNA products in a particularreaction, but comprise some, many, or most of the possiblemutation-containing double-stranded DNA products in a particularreaction. Therefore, a non-comprehensive codon mutagenesis librarycomprises some, many, or most of every possible codon substitution in agene.

The presently disclosed methods can be efficiently used to make alibrary of the desired single mutations when performing the reaction atlow molar ratio of oligonucleotide to DNA template. Increasing the ratioallows one to make libraries comprising mutants with two or more of theoligonucleotide-prescribed mutations. As the ratio of oligonucleotide toDNA template is increased, the distribution of multiple mutations willincrease. In some embodiments, increasing the ratio of oligonucleotideto DNA template will move the distribution from 1, 2, 3, 4, etc.mutations per target nucleic acid molecule (template), for example, to2, 3, 4, 5, etc. mutations per target nucleic acid molecule. The numberof mutations made per target nucleic acid molecule and the number oftarget nucleic acid molecules with that number of mutations depends onthe ratio of oligonucleotide to DNA template. Therefore, the molar ratioof oligonucleotide to DNA template can be varied using the presentlydisclosed methods to control the number of mutations made in a DNAtemplate. As is known in the art, the actual amount of oligonucleotidedesired in the reaction will depend on the nucleic acid sequence of thetemplate and oligonucleotide, the volume of the reaction, the buffersused, the amount of time the reaction is allowed to proceed, and thelike.

The ability of the presently disclosed methods to access mutations (andcombinations of mutations) effectively inaccessible by the current stateof the art should be useful for many fields, such as for the directedevolution of proteins. Furthermore, the ability to custom tailor the setof mutations facilitates constructing focused libraries computationallyenriched for mutants with improved function.

The presently disclosed methods utilizing a single-stranded target DNAmolecule were used to create a comprehensive codon mutagenesis libraryof the TEM-1 β-lactamase gene (described herein below). This library wasdesigned to contain 18,081 members, one for each possible codonsubstitution in the gene (287 positions in TEM-1×63 possible codonsubstitutions). Deep sequencing revealed that approximately 97% of thedesigned single codon substitutions are present in the library.Non-comprehensive or comprehensive codon mutagenesis libraries can becreated using both single-stranded and double-stranded DNA molecules.

D. Kits for Performing PFunkel Mutagenesis

The presently disclosed subject matter also relates to kits forpracticing the methods of the presently disclosed subject matter. Ingeneral, a presently disclosed kit contains some or all of thecomponents, reagents, supplies, and the like to practice a methodaccording to the presently disclosed subject matter. In someembodiments, the term “kit” refers to any intended any article ofmanufacture (e.g., a package or a container) comprising anoligonucleotide and a set of particular instructions for practicing themethods of the presently disclosed subject matter. In other embodiments,the kit comprises a thermostable DNA polymerase and DNA ligase alongwith a set of particular instructions for practicing the methods of thepresently disclosed subject matter. The kit typically comprises aneffective amount of reagents to perform at least one mutagenesisreaction.

In some embodiments, the presently disclosed subject matter provides akit comprising a set of instructions for performing the presentlydisclosed methods. In other embodiments, the kit further comprises athermostable DNA ligase, a thermostable DNA polymerase, and/or anoligonucleotide.

II. DEFINITIONS

The term “nucleic acid molecule” is used broadly herein to mean asequence of deoxyribonucleotides or ribonucleotides that are linkedtogether by a phosphodiester bond. “Nucleic acid molecule” is meant toinclude DNA and RNA, which can be single stranded or double stranded, aswell as DNA/RNA hybrids. Furthermore, the term “nucleic acid molecule”as used herein includes naturally occurring nucleic acid molecules,which can be isolated from a cell, for example, a particular gene ofinterest, as well as synthetic molecules, which can be prepared, forexample, by methods of chemical synthesis or by enzymatic methods suchas by the polymerase chain reaction (PCR), and, in various embodiments,can contain nucleotide analogs or a backbone bond other than aphosphodiester bond.

The terms “polynucleotide” and “oligonucleotide” also are used herein torefer to nucleic acid molecules. Although no specific distinction fromeach other or from “nucleic acid molecule” is intended by the use ofthese terms, the term “polynucleotide” is used generally in reference toa nucleic acid molecule that encodes a polypeptide, or a peptide portionthereof, whereas the term “oligonucleotide” is used generally inreference to a nucleotide sequence useful as a probe, a PCR primer, anantisense molecule, or the like. Of course, it will be recognized thatan “oligonucleotide” also can encode a peptide. As such, the differentterms are used primarily for convenience of discussion.

A polynucleotide or oligonucleotide comprising naturally occurringnucleotides and phosphodiester bonds can be chemically synthesized orcan be produced using recombinant DNA methods, using an appropriatepolynucleotide as a template. In comparison, a polynucleotide comprisingnucleotide analogs or covalent bonds other than phosphodiester bondsgenerally will be chemically synthesized, although an enzyme such as T7polymerase can incorporate certain types of nucleotide analogs into apolynucleotide and, therefore, can be used to produce such apolynucleotide recombinantly from an appropriate template.

The terms “target nucleic acid molecule” or “target DNA molecule” areused herein to refer to the nucleic acid or DNA sequence that ismutagenized using the presently disclosed methods.

The term “mutation” is used herein to refer to an alteration in apolynucleotide sequence. A mutation according to the presently disclosedsubject matter can involve substitution, insertion or deletion. Apolynucleotide in which a mutation has occurred is called a “mutant”.Mutation may be introduced to one or both strands of a double-strandedpolynucleotide. The strand of a double-stranded polynucleotide in whicha mutation has occurred is referred to as a “mutant strand”; the strandwith no mutation introduced is called a “non-mutant strand”. The term“mutagenesis” according to the invention refers to the introduction ofmutations into a polynucleotide sequence. Mutations are preferablyintroduced into a target DNA molecule using one or more mutagenicprimers in an amplification reaction. During the amplification reaction,multiple copies of the strand complementary to the target DNA strand aresynthesized by incorporating the mutagenic primer and extending theincorporated primer using the target strand as a template.

The term “introducing one or more mutations to a single-stranded targetnucleic acid molecule” or “introducing one or more mutations to adouble-stranded target nucleic acid molecule” refers to introducing theone or more mutations into the same copy of the complementary strandsynthesized during the amplification reaction. In addition, “introducingone or more mutations” may also refer to introducing one or moremutations into two or more different copies of the complementary strandssynthesized during the amplification reaction.

The term “substitution” refers to a replacement of one or morenucleotides by different nucleotides. “Insertion” refers to a change innucleotide sequence wherein one or more nucleotides have been added.“Deletion” refers to a change in nucleotide sequence wherein one or morenucleotides are removed.

The term “primer” refers to a polynucleotide, i.e., a purifiedrestriction fragment or a synthetic polynucleotide, that is capable ofacting as a point of initiation of synthesis when placed underconditions in which synthesis of a primer extension productcomplementary to a polynucleotide strand (the “template”) is induced,i.e., in the presence of nucleotides and an agent for polymerizationsuch as DNA polymerase and at a suitable temperature and pH.

The term “mutagenic oligonucleotide” or “mutagenic primer” or “primer”refers to an oligonucleotide used in an amplification reaction, whereinthe oligonucleotide does not precisely match the target hybridizationsequence (e.g., the sequence of a target nucleic acid molecule). Themismatched nucleotides in the mutagenic oligonucleotide are referred toas “mutation site” or “site” with respect to the target sequence (e.g.,the sequence of a target DNA molecule). Thus, during the amplificationreaction, the mismatched nucleotides of the oligonucleotide areincorporated into the amplified product thereby resulting in thesynthesis of a mutant DNA strand comprising the mutagenicoligonucleotide that was used to prime synthesis of the target sequence.A mutagenic oligonucleotide, according to the presently disclosedsubject matter, is complementary to one strand of a target nucleic acidmolecule and contains at least 50%, and preferably at least 75%, atleast 90% of the nucleotide residues capable of base pairing with atarget nucleic acid molecule (e.g., a target DNA molecule).

A “mutagenic oligonucleotide” of the presently disclosed subject matter,also refers to a “degenerate oligonucleotide”. As used herein, a“degenerate oligonucleotide” is a primer mixture synthesized with mixedbases where there is more than one nucleotide sequence possibility forat least one codon coding for an amino acid. An amino acid is coded bythree sequential nucleotides (a codon) in a polynucleotide sequence,more than one codon can encode for the same amino acid. A “degenerateoligonucleotide” according to the presently disclosed subject matter maycomprise one or more degenerated codon sequences.

“Complementary” as used herein refers to the broad concept of sequencecomplementarity between regions of two polynucleotide strands or betweentwo regions of the same polynucleotide strand. It is known that anadenine residue of a first polynucleotide region is capable of formingspecific hydrogen bonds (“base pairing”) with a residue of a secondpolynucleotide region which is antiparallel to the first region if theresidue is thymine or uracil. Similarly, it is known that a cytosineresidue of a first polynucleotide strand is capable of base pairing witha residue of a second polynucleotide strand which is antiparallel to thefirst strand if the residue is guanine. A first region of apolynucleotide is complementary to a second region of the same or adifferent polynucleotide if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region. Afirst polynucleotide that is 100% complementary to a secondpolynucleotide forms a base pair at every nucleotide position. A firstpolynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70%complementary) contains mismatched nucleotides at one or more nucleotidepositions.

“Annealing” as used herein refers to the formation of a double-strandedpolynucleotide between two separate single strands sufficient to primeDNA synthesis in an amplification reaction. “Annealing” occurs throughcomplementary base pairing between the two separated strands, which areat least 50% or more (e.g., 60%, 70%, 80%, 90%, 95% or more)complementary to each other.

“Amplification” as used herein refers to any in vitro method forsynthesizing one or both strands of a polynucleotide template sequence(e.g., a target nucleic acid molecule) with the use of a polymerase.Polynucleotide amplification results in the incorporation of nucleotidesinto a polynucleotide (e.g., DNA) molecule or primer thereby forming anew polynucleotide molecule complementary to the polynucleotidetemplate. The formed polynucleotide molecule and its template can beused as templates to synthesize additional polynucleotide molecules. Asused herein, one amplification reaction may comprise many cycles ofpolynucleotide synthesis. Amplification reactions include the polymerasechain reaction, ligase chain reaction (LCR), and polynucleotide sequencebased amplification, for example.

“Polymerase chain reaction” or “PCR” refers to an in vitro method foramplifying a specific polynucleotide template sequence. The PCR reactioninvolves a repetitive series of temperature cycles. The reaction mixcomprises dNTPs (each of the four deoxynucleotides dATP, dCTP, dGTP, anddTTP), primers, buffers, DNA polymerase, and target nucleic acidmolecule or template.

“A mutation-containing double-stranded DNA product” or “amutation-containing single-stranded DNA product” refers to the doublestrand and/or single strand polynucleotide population generated with amutagenic oligonucleotide during or at the end of an amplificationreaction. The amplified product, according to the presently disclosedmethods, contains mutations to the original target nucleic acid moleculedue to the incorporation of mutagenic primers in the amplificationreaction.

“Thermostable” as used herein refers to an enzyme which is stable toheat, is heat resistant, and functions at high temperatures, e.g., 50°C. to 90° C. Thermostable enzymes are usually purified from thehyperthermophiles of archaebacteria, which grow optimally attemperatures around 100° C.

“Ligating” or “ligation” as used herein refers to covalently attachingpolynucleotide sequences together to form a single sequence. This istypically performed by treatment with a ligase which catalyzes theformation of a phosphodiester bond between the 5′ end of one sequenceand the 3′ end of the other. The ligase catalyses the formation of aphosphodiester bond at the site of a single-stranded break in duplexDNA.

“Mutational efficiency” as used herein refers to the percentage ofpolynucleotide which has incorporated at least one mutagenic primer usedin the amplification reaction.

“Host cell” refers to a cell that comprises a recombinant polynucleotidemolecule, typically a recombinant plasmid or other expression vector.Thus, for example, host cells can express genes that are not foundwithin the native (non-recombinant) form of the cell. The host cell maybe prokaryotic or eukaryotic, including bacterial, mammalian, yeast,aspergillus, and insect cells.

The term “transform” as used herein refers to a process of introducingone or more exogenous DNA molecules into a host cell and/or theexpression of the DNA molecules in the host cell. A host cell with oneor more exogenous DNA molecules is a transformant.

“Elevated temperature” as used herein refers to a temperature above roomtemperature. Generally, “elevated temperature” refers to a temperaturefrom about 30° C. to above 100° C.

“Degrading” a molecule as used herein refers to breaking down themolecule. For example, uracil-containing DNA can be degraded whentransformed into a host cell.

The terms “promoter”, “promoter region” or “promoter sequence” refergenerally to transcriptional regulatory regions of a gene, which may befound at the 5′ or 3′ side of the coding region, or within the codingregion, or within introns. Typically, a promoter is a DNA regulatoryregion capable of binding RNA polymerase in a cell and initiatingtranscription of a downstream (3′ direction) coding sequence. Thetypical 5′ promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence is a transcription initiation site as well as protein bindingdomains (consensus sequences) responsible for the binding of RNApolymerase.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Methods and Materials

Materials

All enzymes were obtained from New England Biolabs (NEB; Ipswich, Mass.,USA) except PfuTurbo Cx Hotstart DNA polymerase, which was obtained fromAgilent Technologies (Santa Clara, Calif., USA). E. coli strain CJ236and NEB 5-alpha F′I^(q) competent cells were obtained from NEB (Ipswich,Mass., USA) and strain DH5α was obtained from Invitrogen (Carlsbad,Calif., USA). R408 helper phage was obtained from Promega (Fitchburg,Wis., USA). All oligonucleotides were ordered from Integrated DNATechnologies (Coralville, Iowa, USA). For the construction of libraryCCM-1 (machine-mixed degenerate oligonucleotides), oligonucleotides wereordered in 96-well format at the 10 nmol synthesis scale such that eacholigonucleotide was provided at a concentration of 100 μM in DI water.For the construction of library CCM-2 (hand-mixed degenerateoligonucleotides), oligonucleotides were ordered in 96-well format atthe 100 nmole synthesis scale such that each oligonucleotide wasprovided at a concentration of 100 μM in DI water. The secondaryoligonucleotide P320, P-gcagaaattcgaaagcaaattcgac (SEQ ID No: 1), wasordered with 5′ phosphorylation. All other chemical reagents wereobtained from Sigma-Aldrich (St. Louis, Mo., USA).

Preparation of CJ236 Competent Cells

E. coli strain CJ236 was plated on LB-agar plates with 15 μg/mLchloramphenicol (Cm), 125 μg/mL deoxythymidine (dThd) and grown at 30°C. Although it is usual to proceed with competent cell preparation froma single colony (especially if using a new cell stock validated by themanufacturer), the desired strain phenotype was first confirmed as anoptional step. A colony with the proper temperature-sensitive dut-1phenotype was identified by replica plating on M9 minimal media agar(Sambrook et al., 2001) supplemented with and without 125 μg/mL dThd andincubated at 30° C. and 42° C. for approximately 40 hours. A colony wasselected which displayed the desired phenotype of stunted growth at 42°C., which was improved with dThd. This colony was used to preparechemically competent cells (Chung et al., 1989). To prevent geneticdrift and reversal of the dut-1 ung-1 phenotype, it is best to propagateCJ236 at 30° C. or lower in deoxythymidine (dThd) supplemented media.These conditions reduce uracil incorporation in DNA (an unfavorablemutagenic event leading to reversions of this phenotype) since uracilincorporation is unnecessary when propagating the strain. However,during preparation of uracil-containing ssDNA or dsDNA template, thestrain was grown at 37° C. without dThd for increased uracilincorporation.

Preparation of Uracil-Containing ssDNA Template

pSkunk3-BLA is a 4.4 kB phagemid derived from pDIM-C8-BLA (Sohka et al.,2009) in which the coding sequence of the Cm resistance gene wasreplaced with the streptomycin/spectinomycin (Sm/Spec) resistance gene.This phagemid was used to transform CJ236 cells which were then platedon LB-agar with 50 μg/mL Spec, 15 μg/mL Cm, and 125 μg/mL dThd andincubated overnight at 30° C. A single colony was used to inoculate 10mL of LB supplemented with Cm, Spec, and dThd as above, which wasincubated with shaking at 30° C. overnight. The cell density of theculture was determined from the OD_(600 nm) using the correlation 2×10⁸CFU/mL-OD_(600 nm). In a 20 mL test tube, 2 mL of TBG media (Trower,1994) with 50 μg/mL Spec was inoculated with 2×10⁷ CFU from theovernight culture and 1×10⁸ pfu R408 helper phage for a multiplicity ofinfection (MOI) of 5. This culture was incubated for 6 hours at 37° C.with shaking at 300 rpm. The culture was then centrifuged for 5 minutesat 16,100×g to pellet the cells, and the phage-containing supernatantrecovered. Then 300 μL of 2.5 M NaCl/20% PEG was added to thesupernatant and the mixture was incubated at 4° C. for 1 hour toprecipitate the phage. The phage was pelleted by centrifugation at20,817×g for 10 minutes at 4° C. The liquid supernatant was discardedand the phage pellet resuspended in 150 μL PBS. The Qiagen QIAprep SpinM13 kit (#27704; Hilden, Germany) was then used to purify ssDNA from thephage as per the manufacturer's directions. The absorbance at 260 nm ofthe ssDNA sample was measured using a Nanodrop ND-1000 spectrophotometer(Thermo Fisher Scientific, Waltham, Mass., USA) and converted to aconcentration using the relation 1.0 A_(260 nm)=33 ng/μL.

Site-Directed PFunkel Mutagenesis Using a ssDNA Template

All steps were performed in a pre-programmed Eppendorf Mastercyclerpersonal thermocycler (Eppendorf, Hamburg, Germany). A mutagenicoligonucleotide (5′-gacaccacgatgcatgcagcaatggc; SEQ ID No: 2) encoding ac542a mutation in the bla gene was phosphorylated in a 50 μL reactioncontaining 1×T4 PNK buffer, 1 mM ATP, 5 mM DTT, 3.0 μM oligonucleotideand 10 units T4 PNK. The reaction was incubated at 37° C. for 1 hour,and the enzyme inactivated at 65° C. for 20 minutes.

The PFunkel reaction was prepared in a 0.5 mL eppendorf tube containing1× pfuTurbo Cx hotstart DNA polymerase buffer, 10 mM DTT, 0.5 mM NAD+,0.2 mM dNTPs, 1 μL of the kinase reaction, 1 μg (0.75 pmol) of dU-ssDNAtemplate, 2.5 units PfuTurbo Cx hotstart DNA polymerase, and 200cohesive end units Taq ligase in a total volume of 100 μL. The free Mg2+concentration should be maintained between 0.5-2.5 mM since lowconcentration reduces polymerase fidelity while high concentration leadsto nonspecific annealing of oligonucleotides (PfuTurbo Cx hotstart DNApolymerase Instruction Manual, 2009). The volume of kinase reactionadded should therefore be minimized to maintain Mg2+ concentration inthe mutagenesis reaction close to the 2 mM Mg2+ provided in the 1×polymerase buffer.

The following denaturation/annealing/extension/ligation steps wereperformed: 95° C. for 3 min, 55° C. for 90 sec, 68° C. for 15 min and45° C. for 15 min. Then 3.8 pmol of oligonucleotide P320(5′-P-gcagaaattcgaaagcaaattcgac; SEQ ID No: 1) was added and one morecycle of 95° C. for 30 sec, 55° C. for 45 sec, 68° C. for 10 min and 45°C. for 15 mins was performed. Then 10 units of UDG and 30 units ofExoIII were added and incubated at 37° C. for 1 hr followed by aninactivation step at 70° C. for 20 min.

Five μL of the unpurified reaction was used to directly transform 100 μLof DH5α chemically competent cells (Chung et al., 1989). The entiretransformation was plated on an LB-agar plate with 50 μg/mL Spec andincubated overnight at 37° C. To obtain more transformants, theremaining DNA was then purified using the Zymo DNA Clean & Concentratorkit (Zymo Research Corporation, Irvine, Calif., USA) according to themanufacturer's instructions and eluted in 15 μL of 1×EB. One μL waselectroporated into 50 μL DH5α electrocompetent cells and then incubatedwith SOC recovery media for 1 hr at 37° C. with shaking at 250 rpm. Thetransformation was plated on LB-agar with 50 μg/mL Spec and incubatedovernight at 37° C.

For the experiments of Table 2, the reaction was scaled down to 200 ngtemplate and 20 μL volume.

Multi-Site PFunkel Mutagenesis Using a ssDNA Template

Four oligonucleotides were designed to introduce NNN random bases atcodon positions 42, 104, 182, and 238 in the bla gene with respectivesequences: 5′-gatcagttgggtnnncgagtgggttac (SEQ ID No: 3),5′-gaatgacttggttnnntactcaccagtcac (SEQ ID No: 4),5′-cgtgacaccacgnnncctgcagcaatg (SEQ ID No. 5),5′-aaatctggagccnnngagcgtgggtct (SEQ ID No. 6). These oligos werecombined in equimolar amounts and phosphorylated in a 50 μL reactioncontaining 1×T4 PNK buffer, 1 mM ATP, 5 mM DTT, 6.0 μM totaloligonucleotide and 10 units T4 PNK. The reaction was incubated at 37°C. for 1 hour, and the enzyme inactivated at 65° C. for 20 minutes.

The annealing reaction was prepared in a 0.5 mL eppendorf tubecontaining 1× pfuTurbo Cx hotstart DNA polymerase buffer, 2 μL of kinasereaction, and 1 μg of pSkunk3-bla ssDNA template in a total volume of 77μL. The annealing was performed by heating to 95° C. for 3 min, then 55°C. for 10 min, and holding at 55° C.

Meanwhile, in a separate PCR tube, 1×PfuTurbo Cx hotstart DNA polymerasebuffer and 2.75 units of PfuTurbo Cx hotstart DNA polymerase werecombined in a total volume of 5.5 μL. The hotstart polymerase was heatactivated by heating to 95° C. for 3 min.

After the annealing step, 10 mM DTT, 1 mM NAD+, 0.2 mM dNTPs, 5 μL ofthe activated polymerase solution, and 200 cohesive end units Taq ligasewas added bringing the total volume to 100 μL. The reaction was mixed byslowly and gently pipetting up and down. Extension and ligation of themutant strand was performed at 65° C. for 15 min and 45° C. for 15 min.Five units of UDG and 2 units of ExoIII were added and the mixture wasincubated at 37° C. for 1 hr followed by an inactivation step at 70° C.for 20 min. A total of 3.8 pmol of oligonucleotide P320 was added andone more cycle of 95° C. for 30 sec, 55° C. for 45 sec, 68° C. for 10min and 45° C. for 15 mins was performed. The DNA was purified using theZymo DNA Clean & Concentrator kit (Zymo Research Corporation, Irvine,Calif., USA) according to the manufacturer's instructions and eluted in15 μL of DI water. This solution was vacuum concentrated down to 1-2 μL,electroporated into 50 μL DH5α electrocompetent cells and then incubatedwith SOC recovery media for 1 hr at 37° C. with shaking at 250 rpm. Thetransformation was plated on LB-agar with 50 μg/mL Spec and incubatedovernight at 37° C.

For the experiments of Table 2, the reaction was scaled down to 200 ngtemplate and 20 μL volume.

Comprehensive Codon Mutagenesis by PFunkel Using a ssDNA Template

All steps were performed in a pre-programmed Eppendorf Mastercyclerpersonal thermocycler. Equimolar amounts of 287 different mutagenicoligos were combined in a single tube at a total oligonucleotideconcentration of 100 μM. The oligonucleotides were phosphorylated in a50 μL reaction containing 1×T4 PNK buffer, 1 mM ATP, 5 mM DTT, 0.038 μMoligonucleotides and 10 units T4 PNK. The reaction was incubated at 37°C. for 1 hour, and the enzyme inactivated at 65° C. for 20 minutes.

The PFunkel reaction was prepared in a 0.5 mL eppendorf tube containing1×PfuTurbo Cx hotstart DNA polymerase buffer, 10 mM DTT, 0.5 mM NAD+,0.2 mM dNTPs, 1 μL of the kinase reaction, 1 μg (0.75 pmol) of dU-ssDNAtemplate, 2.5 units PfuTurbo Cx hotstart DNA polymerase, and 200cohesive end units Taq ligase in a total volume of 100 μL. The followingdenaturation/annealing/extension steps were performed: 95° C. for 2 min,15 cycles of 95° C. for 30 sec, 55° C. for 45 sec, and 68° C. for 6.5min. At the 95° C. step of cycles 6 and 11, 1 μL of the kinase reactionwas added and mixed in by stirring with the pipette tip. The reactionwas then incubated at 45° C. for 15 min for ligation to occur. Then 3.8pmol of oligonucleotide P320 (5:1 molar ratio oligonucleotide totemplate) was added and one more cycle of 95° C. for 30 sec, 55° C. for45 sec, and 68° C. for 10 min was carried out. The reaction was againincubated at 45° C. for 15 min. Then 10 units of UDG and 30 units ofExoIII were added and incubated at 37° C. for 1 hr followed by aninactivation step at 70° C. for 20 min. The DNA was then purified usingthe Zymo DNA Clean & Concentrator kit (Zymo Research Corporation,Irvine, Calif., USA) according to the manufacturer's instructions andeluted in 15 μL of DI water. This volume was then vacuum concentrateddown to 1-2 μL. For CCM-1, the DNA was electroporated into 50 μL DH5αelectrocompetent cells and then incubated with SOC recovery media for 1hr at 37° C. with shaking at 250 rpm. The entire volume was then platedon a Nalgene Bioassay dish (D4803; 245 mm×245 mm×25 mm) containingLB-agar with 50 μg/mL Spec and incubated overnight at 37° C. For CCM-2,the DNA was used to transform NEB 5-alpha F′Iq competent cells as perthe manufacturer's instructions, and then plated on a Nalgene Bioassaydish containing LBagar with 50 μg/mL Spec, 15 μg/mL tetracycline, and 2w/v % glucose.

Reaction Conditions for the PFunkel Mutagenesis Using a ssDNA Template

Table 1 shows representative embodiments of reaction conditions for thePFunkel mutagenesis using a single-stranded DNA template.

TABLE 1 Reaction conditions for PFunkel using a ssDNA template.Comprehensive codon Site-directed mutagenesis Multiple-site mutagenesismutagenesis Start with PfuTurbo Cx buffer PfuTurbo Cx buffer PfuTurbo Cxbuffer 2.5 units PfuTurbo Cx 1 μg (0.75 pmol) of dU- 2.5 units PfuTurboCx polymerase ssDNA polymerase 10 mM DTT 2 μL, of 6 μM kinased 10 mM DTT0.5 mM NAD⁺ mutagenic oligo mixture 0.5 mM NAD⁺ 0.2 mM dNTPs 77 μL,total volume 0.2 mM dNTPs 1 μg (0.75 pmol) of dU- 1 μg (0.75 pmol) ofdU- ssDNA ssDNA 1 μL, of 3 μM kinased 1 μL, of 0.038 μM kinasedmutagenic oligo mutagenic oligo mix 200 cohesive end units Taq 200cohesive end units Taq ligase ligase 100 μL, total volume 100 μL, totalvolume Initial step none None 95° C. for 2 min. Cycling 1 cycle of 95°C. for 3 min, 15 cycles of 95° C. for 3 min 55° C. for 10 min 95° C. for30 sec 55° C. for 90 sec hold at 55° C. 55° C. for 45 sec 68° C. for 15min add 68° C. for 6.5 min. 10 mM DTT At the 95° C. step of cycles 6 0.5mM NAD⁺ and 11, an additional 1 μL, 0.2 mM dNTPs of the kinase reactionwas 2.5 units PfuTurbo Cx added. (previously heat activated) 200cohesive end units Taq ligase bringing the total volume to 100 μL 65° C.for 15 min Ligation 45° C. for 15 min add Add 3.8 pmol of kinased oligoP320 (5:1 molar ratio oligo to template) Synthesis one cycle of ofsecond 95° C. for 30 sec strand 55° C. for 45 sec containing 68° C. for10 min mutation 45° C. for 15 min (ligation) Degradation Shift to 37° C.Shift to 37° C. Shift to 37° C. of Add 10 units of UDG + 30 Add 5 unitsof UDG + 2 Add 10 units of UDG + 30 template units of ExoIII; incubate 1units of ExoIII; incubate 30 min units of ExoIII; incubate 1 and side-hour at 37° C.; 70° C. for 20 min at 37° C.; 70° C. for 20 min hour at37° C.; 70° C. for 20 min products (heat inactivation) (heatinactivation) (heat inactivation) Final step Purify DNA (optional stepto increase number of transformants) and transform454 GS FLX High-Throughput Sequencing

Transformants were recovered from agar plates with LB broth, and plasmidDNA recovered using the Qiagen QIAprep Spin Miniprep kit (27106). Theplasmid DNA was linearized by restriction endonuclease digestion withNdeI. PCR amplicons of each of the three bla libraries were createdusing Titanium Lib-A fusion primers that included a 10-base MID barcode.Each 25 μL PCR reaction had 1-2 ng linearized template DNA, 0.4 μM eachprimer, 200 μM each dNTP, 1×HF Phusion buffer, and 2 units Phusionhigh-fidelity polymerase. Cycler conditions were 98° C. for 30 sec, 30cycles of 98° C. for 30 sec, 55° C. for 30 sec, 72° C. for 30 sec, andthen 72° C. for 5 min. PCR products were visualized on an ethidiumbromide 1% agarose gel, and then gel purified using the QIAquick GelExtraction Kit (28706). Amplicons were furthered purified using theAgencourt AMPure XP PCR Purification kit (A63880; Beckman Coulter, Brea,Calif., USA), to remove short DNA fragments, primers, and primer dimers.DNA concentration was determined using the Quant-iT Picogreen dsDNAAssay kit (P7589; Life Technologies, Carlsbad, Calif., USA). Ampliconsfrom each sub-library were diluted to 1E9 molecules/μL in 1×TE, equalvolumes pooled together and then further diluted to 1E7 molecules/μL inDI water. 454 sequencing was performed by Tufts University Core Facilityon a Roche 454 GS FLX+ instrument (Basel, Switzerland). The sequencingdata was then analyzed using the Galaxy open web-based platform (Goeckset al., 2010; Blankenberg et al., 2010; Giardine et al., 2005) andcustom Matlab scripts.

Identification of Adaptive Codon Substitutions for Tazobactam Resistancein TEM-1

Library CCM-2 was plated at a density of about 500 CFU/cm²(non-selective conditions) on LB-agar plates supplemented with 50 μg/mLSpec, 300 μM IPTG, 100 μg/mL ampicillin and 4 μg/mL or 6 μg/mLtazobactam. Plates were incubated at 37° C. for 17 hours. The tazobactamconcentration chosen was 1.3 or 2-fold higher than the concentration atwhich cells bearing wildtype TEM-1 could grow effectively. Largecolonies on the plates were chosen at random for sequencing.

Selected single base mutations were re-introduced into TEM-1 bysite-directed PFunkel mutagenesis on the 20 μL volume scale. The MIC forampicillin and piperacillin of the mutants was assessed with and without6 μg/mL tazobactam by spotting 104 CFU on Mueller-Hinton agar platescontaining 50 μg/mL Spec, 300 μM IPTG, and √2-fold increments of eitherampicillin or piperacillin. Plates were incubated at 37° C. for 12 hrs.

Preparation of Uracil-Containing dsDNA Template for Phage-Less PFunkel

A 10 mL LB culture of CJ236 cells with the pSkunk3-bla plasmid wasincubated overnight at 37° C. with shaking at 250 rpm. Plasmid dU-dsDNAwas then isolated using the Qiagen QIAprep Spin Miniprep kit (#27106;Qiagen, Hilden, Germany) and the concentration quantified using aNanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham,Mass., USA).

Site-Directed PFunkel Mutagenesis Using a Plasmid dsDNA Template

All steps were performed in a pre-programmed Eppendorf Mastercyclerpersonal thermocycler (Eppendorf, Hamburg, Germany). A mutagenic oligo(5′-gacaccacgatgcatgcagcaatggc) encoding a c542a mutation in TEM-1 wasphosphorylated in a 50 μL reaction containing 1×T4 PNK buffer, 1 mM ATP,5 mM DTT, 1.5 μM oligo and 10 units T4 PNK. The reaction was incubatedat 37° C. for 1 hour and the enzyme inactivated at 65° C. for 20minutes.

The PFunkel reaction was prepared in a 0.5 mL tube containing 1×PfuTurbo Cx hotstart DNA polymerase buffer, 10 mM DTT, 0.5 mM NAD+, 0.2mM dNTPs, 1 μL of the kinase reaction, 1 μg (0.38 pmol) of dU-dsDNAtemplate, 2.5 units PfuTurbo Cx hotstart DNA polymerase, and 200cohesive end units Taq ligase in a total volume of 100 μL. The free Mg2+concentration should be maintained between 0.5-2.5 mM since lowconcentration reduces polymerase fidelity while high concentration leadsto nonspecific annealing of oligos (PfuTurbo Cx hotstart DNA PolymeraseInstruction Manual, 2009). The volume of kinase reaction added shouldtherefore be minimized to maintain Mg2+ concentration in the mutagenesisreaction close to the 2 mM Mg2+ provided in the 1× polymerase buffer.The following denaturation/annealing/extension/ligation steps wereperformed: 95° C. for 3 min, 55° C. for 90 sec, 68° C. for 15 min and45° C. for 15 min. Next, 10 units of UDG and 30 units of Exo III wereadded and the reaction was incubated at 37° C. for 1 hr followed by aninactivation step at 70° C. for 20 min. A total of 3.8 pmol of oligoP320 (5′-Pgcagaaattcgaaagcaaattcgac; SEQ ID No: 1) was added and onemore cycle of 95° C. for 30 sec, 55° C. for 45 sec, 68° C. for 10 minand 45° C. for 15 min was performed. The DNA was purified using the ZymoDNA Clean & Concentrator kit (Zymo Research Corporation, Irvine, Calif.,USA) according to the manufacturer's instructions and eluted in 15 μL ofDI water. This solution was vacuum concentrated down to 1-2 μL,electroporated into 50 μL DH5α electrocompetent cells, which wereincubated with SOC recovery media for 1 hr at 37° C. with shaking at 250rpm. The transformation was plated on LB-agar with 50 μg/mL Spec andincubated overnight at 37° C.

Multi-Site PFunkel Mutagenesis Using a Plasmid dsDNA Template

Four oligos were designed to introduce the four mutations A42G, E104K,M182T, and G238S in the bla gene with respective sequences:5′-gatcagttgggtgga cgagtgggttac (SEQ ID No: 7),5′-ctcagaatgacttggttaagtactcaccagtcacag (SEQ ID No: 8),5′-gtgacaccacgacgcctgcagcaatggcaacaac (SEQ ID No: 9),5′-gctgataaatctggagccagtgagcgtgggtctcg (SEQ ID No: 10). These oligoswere combined in equimolar amounts and phosphorylated in a 50 μLreaction containing 1×T4 PNK buffer, 1 mM ATP, 5 mM DTT, 3 μM totaloligo and 10 units T4 PNK. The reaction was incubated at 37° C. for 1hour and the enzyme inactivated at 65° C. for 20 minutes.

The annealing reaction was prepared in a 0.5 mL eppendorf tubecontaining 1× PfuTurbo Cx hotstart DNA polymerase buffer, 2 μL of kinasereaction, and 1 μg of dUdsDNA template in a total volume of 77 μL. Theannealing was performed by heating to 95° C. for 3 min, then 55° C. for10 min, and holding at 55° C.

Meanwhile, in a separate PCR tube, 1×PfuTurbo Cx hotstart DNA polymerasebuffer and 2.75 units of PfuTurbo Cx hotstart DNA polymerase polymerasewere combined in a total volume of 5.5 μL. The hotstart polymerase washeat activated by heating to 95° C. for 3 min.

After the annealing step, 10 mM DTT, 0.5 mM NAD+, 0.2 mM dNTPs, 5 μL ofthe activated polymerase solution, and 200 cohesive end units Taq ligasewas added bringing the total volume to 100 μL. The reaction was mixed byslowly and gently pipetting up and down. Extension and ligation of themutant strand was performed at 65° C. for 15 min and 45° C. for 15 min.Five units of UDG and 2 units of ExoIII were added and the mixture wasincubated at 37° C. for 1 hr followed by an inactivation step at 70° C.for 20 min. A total of 3.8 pmol of oligo P320 was added and one morecycle of 95° C. for 30 sec, 55° C. for 45 sec, 68° C. for 10 min and 45°C. for 15 min was performed. The DNA was purified using the Zymo DNAClean & Concentrator kit (Zymo Research Corporation, Irvine, Calif.,USA) according to the manufacturer's instructions and eluted in 15 μL ofDI water. This solution was vacuum concentrated down to 1 μL-2 μL,electroporated into 50 μL DH5α electrocompetent cells and then incubatedwith SOC recovery media for 1 hr at 37° C. with shaking at 250 rpm. Thetransformation was plated on LB-agar with 50 μg/mL Spec and incubatedovernight at 37° C.

Example 2 Site-Directed PFunkel Mutagenesis

A mutagenic oligonucleotide encoding a c542a (P183H in the protein)mutation in the TEM-1 β-lactamase (TEM-1) gene was first 5′phosphorylated in a kinase reaction. The phosphorylated oligonucleotidewas then combined with the ssDNA uracil-containing template in molarratio of 4:1 together with the polymerase and ligase. The incubationtemperatures were cycled to perform a denaturing, annealing, extension,and ligation step to complete the mutated second strand and seal thenick. A second primer that annealed to the new strand outside the genewas added to the reaction, and the denaturing, annealing, extension, andligation steps were repeated. Exo III and UDG were then added to thereaction to remove the template and undesired side-products. All stepsfor this procedure took about 3 hrs to complete.

A DNA gel showing steps in the reaction and various control reactions isshown in FIG. 2. The white triangle indicates the appearance of a bandat the correct size to be phagemid cccDNA. Without being bound to anyone particular theory, it is believed that the amount of cccDNA productin the complete reaction prior to transformation (lane 8) is too low tovisualize.

A transformation of 5 μL of the unpurified reaction with 100 μL ofchemically competent cells yielded over 1000 transformants, illustratingthat, in some embodiments, DNA purification is not necessary when usingthe presently disclosed methods. The remaining DNA was purified using aspin column and 1/15th of the product was electroporated intoelectrocompetent DH5α E. coli yielding 533,000 transformants Sequencingof the TEM-1 gene from 23 colonies showed that all 23 (100%) containedthe c542a mutation encoded by the oligonucleotide. No undesiredmutations were

observed. The high mutational efficiency of the presently disclosedmethods was further substantiated using eleven differentoligonucleotides encoding either a 1 or 2 base substitution at differentlocations of the gene (Table 2).

TABLE 2 Results of additional testing of PFunkel site-directedmutagenesis and multi site mutagenesis using a single-stranded DNA template. Oligonucleotides added  Type of in reaction^(a)Number of PFunkel Mutation(s) Intended correct sequences^(b)Site-directed M69L 2 of 2 Mutagenesis Y105S 2 of 2 Y105D  2 of 2^(c)Y105N 2 of 2 S235T 2 of 2 R244S 2 of 2 N276D 2 of 2 A42G 2 of 2 E104K 2 of 2^(d) M182Q 2 of 2 G238A 2 of 2 Multisite A42G, E104K 2 of 2Mutagenesis A42G, M182Q 2 of 2 A42G, G238A 2 of 2 E104K, M182Q 2 of 2E104K, G238A 1 of 2 M182Q, G238A 1 of 2 A42G, E104K, M182Q 1 of 2A42G, E104K, G238A 1 of 2 A42G, M182Q, G238A 1 of 2 E104K, M182Q, G238A1 of 2 A42G, E104K, M182Q, G238A 2 of 2 ^(a)M69L encodes atg69ctg(5′-cccgaagaacgttttccaatgctgagcacttttaaa-3′; SEQ ID No: 11) Y105Sencodes tac105tcc (5′-tgacttggttgagtcctcaccagtcacaga-3′; SEQ ID No: 12)Y105D encodes tac105gac (5′-tgacttggttgaggactcaccagtcacaga-3′; SEQ IDNo: 13) Y105N encodes tac105acc (5′-tgacttggttgagaactcaccagtcacaga-3′;SEQ ID No: 14) S235T encodes tct235act(5′-attgctgataaaactggagccggtgagc-3′; SEQ ID No: 15) R2445 encodescgc244agc (5′-gagcgtgggtctagcggtatcattgca-3′; SEQ ID No: 16) N276Dencodes aat276gat (5′-atggatgaacgagatagacagatcgctgaga-3′; SEQ ID No: 17)A42G encodes gca42ggg (5′-gatcagttgggtgggcgagtgggttac-3′; SEQ ID No: 18)E104K encodes gag104aag (5′-ctcagaatgacttggttaagtactcaccagtcacag-3′; SEQID No: 19) M182Q encodes atg182cag (5′-cgtgacaccacgcagcctgcagcaatg-3′;SEQ ID No: 20) G238A encodes ggt238gca(5′-aaatctggagccgcagagcgtgggtct-3′; SEQ ID No: 21) ^(b)The number ofclones with all intended mutations out of the total number of clonessequenced. ^(c)One clone had an additional, unintended point mutation.^(d)One clone had an additional, unintended mutation found near thedesired mutation, presumably resulting from the synthesized mutagenicoligo possessing a misincorporated base.

Example 3 Multi-Site PFunkel Mutagenesis

To demonstrate multiple-site mutagenesis using PFunkel mutagenesis, fourmutagenic oligonucleotides designed to create site-saturation librariesof four codons in different regions of the TEM-1 gene simultaneouslywere synthesized. The oligonucleotides encoding NNN at codon positionsA42, E104, M182, and G238 were combined with the ssDNA template suchthat each oligonucleotide was present in an oligonucleotide to templatemolar ratio of 4:1. Electroporation of the entire reaction product afterspin column purification yielded 5.8 million transformants. Sequencingof the TEM-1 gene of 10 colonies showed that 7 variants had mutations atall 4 designated codon positions, 2 had mutations at 3 positions, and 1had mutations at 2 positions (Table 3). Twenty-nine of the 35 codonsubstitutions were unique and no undesired mutations were observed.Multi-site PFunkel was further substantiated by constructing 11different double, triple, or quadruple mutants at 73% efficiency (Table2). The error rate for single or multi-site PFunkel mutagenesis wasapproximately 5×10⁻⁵, higher than expected based on the error rate ofPfuTurbo Cx in a PCR reaction.

TABLE 3 Mutations in 10 clones of the naïve multi-site libraryAmbler Position^(a) 42 104 182 238 Amino Amino Amino Amino Colony CodonAcid Codon Acid Codon Acid Codon Acid TEM-1 gca A gag E atg M ggt G  1ggc G — — cag Q tgg W  2 agc S ggg G gga G — —  3 cgg R ggg G cgg R agaR  4 tgc C ggg G agg R cgg R  5 tgc C gcg A ggg G gtg V  6 ggc G tgg Wccg P ctg L  7 ggc G ggg G ttg L gac D  8 ggt G ggg G gca A atc I  9 gcgA ggt G cat H atc I 10 — — — — — — tcc S ^(a)oligo for position 42:5′-gatcagttgggtnnncgagtgggttac-3′; SEQ ID No: 22 oligo for position 104:5′-gaatgacttggttnnntactcaccagtcac-3′; SEQ ID No: 23 oligo for position182: 5′-cgtgacaccacgnnncctgcagcaatg-3′; SEQ ID No: 24 oligo for position238: 5′-aaatctggagccnnngagcgtgggtct-3′; SEQ ID No: 25

Example 4 Comprehensive Codon Mutagenesis

The presently disclosed methods were used to create a library designedto encompass all possible single codon substitutions in the TEM-1 gene(287 codons×63 possible codon substitutions at each codon=18,081 desiredmutants). In this example, it was not desired to create a library withmore than one codon substituted. Such a library is the equivalent ofperforming site-saturation mutagenesis at all positions in the genesimultaneously. At least one advantage of the presently disclosedmethods is that one does not have to perform 287 separate mutagenesisreactions or 287 separate gene syntheses to create this library. Thelibrary would also be much closer to a true random mutagenesis librarythan one created by error prone PCR, which is biased towards certainbase substitutions made by the polymerase and certain amino acidsubstitutions accomplished by single base mutations.

The 287 degenerate mutagenic oligonucleotides (one for each of the 287codons to be mutated) were designed in silico using a Matlab script oralgorithm (FIG. 3). For each gene position to be randomized, thealgorithm scans through many possible oligonucleotides, assigns each ascore based on specific guidelines, and then selects the best scoringoligonucleotide sequence. Published design criteria (QuikChangeSite-Directed Mutagenesis Kit, Instruction Manual) along with earlyexperimental data were used to develop the following oligonucleotidecriteria: (a) the oligonucleotide length can vary from 27 to 40 bases;(b) the mismatched bases must be flanked by greater than or equal to 12bases on each side; (c) the Tm must be greater than or equal to 62° C.;(d) the GC content must be greater than or equal to 40%; (e)oligonucleotides with a stable 5′ end and an unstable 3′ end are favoredto prevent non-specific annealing and extension; and (f)oligonucleotides with polynucleotide repeats, hairpin structures, and apropensity for dimerization are penalized. Each oligonucleotide isdesigned to replace a different codon in the bla gene with a randomsequence (NNN). The script can be easily modified for designing othertypes of libraries.

The oligonucleotides were purchased in desalted 96-well format usingmachine-mixed degenerate bases and pooled. To minimize the occurrence ofmultiple mutations, the total oligonucleotide to ssDNA template ratiowas kept low (1:20), which makes two oligonucleotides annealing to thesame ssDNA template unlikely. To increase the yield and efficiency ofthe reaction, a cycling reaction of denaturing, annealing, and extensionwas implemented to allow multiple chances for each oligonucleotide toproductively anneal. Fifteen cycles were performed with additionaloligonucleotides spiked in at the 6th and 11th cycle. Additional cyclesand oligonucleotide additions can be performed if larger libraries aredesired. Without wishing to be bound to any one particular theory, thepresently disclosed methods are analogous to that of a discontinuous fedbatch reactor—a reaction strategy to minimize undesirable side productsthat occur with a high concentration of one of the reactants (Denbigh,1944).

In some embodiments, the library can be created in a single tube. Inthis example, the library was divided into thirds corresponding to each⅓ of the gene to facilitate characterization of the library by454-GS-FLX Titanium sequencing, which has a read length of approximately400 bp for the sequencing of amplicons from pools of DNA. Transformationof the entire reaction product yielded approximately 500,000transformants for each library.

Sequencing of 30 members of each library indicated that the librariesmostly consisted of single codon substitutions (87%) with the remainderbeing wildtype (13%) (Table 4). No clones with multiple mutations wereobserved. Two of the sequences contained a single mutation outside theregion subjected to mutagenesis, which was attributed to polymeraseerror. It was postulated that additional rounds of cycling and mutagenicoligonucleotide addition would lower the fraction of wild-type sequencescloser to the theoretical minimum of 1.6% (i.e., 1/64 of the NNNcontaining oligonucleotides encode the wildtype codon). These threelibraries collectively were named CCM-1.

The CCM-1 library was extensively analyzed using 454-GS-FLX Titaniumsequencing. FIG. 4 shows the frequency of each of the 63 possible codonsubstitutions for each of the 287 codons of TEM-1, except for the 3% ofthe 18,081 codon substitutions that were not observed. The frequency isbased on 454 sequencing in which 738,615 codon substitutions wereobserved in 787,488 reads. The frequency is normalized to the frequencythat would occur if all substitutions were evenly distributed among the18,081 possible substitutions (i.e., frequency=1.0 means that thesubstitution was observed 738,615/18,081=41 times). The number of codonsubstitutions observed resulting from sequencing errors is small(approximately 4% of the 738,615 codon substitutions observed).

The analysis indicated that 96-97% of the 18,081 desired codonsubstitutions were present in the library and greater than or equal to97% of library members with a codon substitution contained only onecodon substitution (Table 5, described herein below). The frequency ofcodon substitutions observed as a function of gene position showed thata few positions were hotspots for substitutions and that the frequencyof codon substitutions had a broad distribution (FIG. 4). Withoutwishing to be bound to any one particular theory, it is believed thatthe occurrence of hotspots reflected the suitably of the respectiveoligonucleotides for this mutagenesis technique. Codon substitutionswith a single base pair change were observed at about twice the expectedfrequency, and this came at the expense of fewer codon substitutionswith three base pair changes (Table 4). Without wishing to be bound toany one particular theory, it is believed that a portion of the biastowards single base substitutions was likely due to polymerase errorsduring library construction, polymerase errors during the PCR-basedamplicon preparation for sequencing, and 454 sequencing errors, all ofwhich would be primarily single base substitutions. The remainder of thebias may reflect the increased mismatch between the mutagenicoligonucleotide and the template for codon substitutions with threemutated bases. Still, although codon substitutions with three base pairchanges may have been somewhat disfavored, 95% of the 7749 designed3-base pair change codon substitutions in the 454 sequencing resultswere observed (Table 4). Since 454 sequencing errors with three basepair changes in a codon are likely very rare, it is believed most if notall of these 3-base substitutions were present in the library.

454 GS FLX High-Throughput Sequencing Analysis of the ComprehensiveCodon Substitution Library

Barcoded amplicons from the three CCM-1 libraries were created by PCRand pooled. Additionally, barcoded amplicons created from the wildtypeTEM-1 gene were added to the pool as a control for sequencing errors.787,488 reads that passed quality filtering were obtained, with a medianlength of 354 bases. A total of 99% of the reads spanned the entiremutated region of the amplicon. The reads of the library DNA displayed ahigher frequency of both wildtype (26%) and multiple mutations (17%) atthe expense of single codon mutations (57%) as compared to the Sangersequencing of 90 clones. However, this was determined to be an artifactof the amplicon preparation known as “PCR jumping,” a well-documentedoccurrence during PCR amplification of highly-identical, heterogeneoustemplate sequences in which chimera PCR products are produced (Hollandet al., 2011; Meyerhans et al., 1990). This was confirmed to be the caseby Sanger sequencing of 28 individual PCR amplicons of which 36% had nomutations, 50% had one codon mutation, and 14% had multiple codonmutations (Table 4). This closely matched the proportions in the 454sequencing. The sequencing of wild-type TEM-1 indicated that thesequencing error rate (0.035 codon substitutions per read) was much lessthan the frequency of codon substitutions observed in the reads of thelibrary DNA (0.94 per read). It was concluded that 96% of the codonsubstitutions observed in the 454 sequencing reads were present in thelibrary, with the remainder being sequencing errors. Of the codonsubstitutions present in the library, less than or equal to 3% werepresent in library members with multiple mutations (based on Sangersequencing). In the 454 sequencing reads of the library, 97.0% of the18,081 intended codon substitutions were observed at least once. In theworst-case scenario, in which sequencing errors are assigned tomutations with the lowest numbers of occurrences, 84.8% of the 18,081possible mutants were present in the library. If sequencing errors areevenly distributed across all codon substitutions, 96.4% were present.In the best-case scenario, in which errors are assigned to mutants thatare highly represented, 96.8% of the 18,081 mutants were present. It isbelieved that the true coverage of the library lies between 96.4% and97% and likely closer to 97%, since 454 sequencing is known to exhibitsequence-dependent common errors. Among the 72 sequencing errors in thereads of wild-type TEM-1, one particular substitution appeared fivetimes and five codon substitutions appeared twice. More extensivesequencing of wild-type TEM-1 would be

necessary to accurately determine the frequency at which each of the18,081 possible codon substitutions appear because of sequencing error,and thus the true frequency of each codon substitution in the library.

Both the Sanger and the 454 sequencing indicate that G's are present inmutated codons 2.3 times more frequently than any one of the other threebases (Table 5). The high frequency of G's is also apparent in thesequences of naïve members of the multi-site mutagenesis library (Table3), which used four specific primers from the set of 287. Thedistribution of the frequency of the substituted codons stronglyreflects this bias (FIG. 5A) whereas the distribution of the frequencyof codons substituted into does not (FIG. 5B). Since TEM-1 has roughlyan equal frequency of all bases, it is concluded that this bias resultsfrom a 2.3-fold bias for incorporation of G's during the synthesis ofthe machine-mixed degenerate oligonucleotides. This bias contributed tothe underrepresentation of certain mutations, as the frequency of G's incodon substitutions not observed in the 454 sequencing was 0.068. Thefrequency is normalized to that expected if all codon substitutionsoccurred with equal frequency. The codon substitutions are color codedas to the number of G's in the substituted codon. TEM-1 lacks threecodons (TAG, TGA, AGG) so those codons are not included in the codonssubstituted into.

TABLE 4 Statistics of comprehensive codon mutagenesis library CCM-1.Sequencing of Sequencing PCR 454 Expected of individual ampliconssequencing 454 in an ideal clones of the used in 454 of the sequencinglibrary library sequencing library of TEM-1 Sequences 90 clones 28clones 787,488 2040 reads reads Percent of reads that cover   100%  100% 98.95% 99.75% entire gene segment Number of mutated codons in78 + 2^(a) 22 738,615 72 all sequences Mean mutated codons per 0.98440.87 0.79 0.94 0.035 sequence Percent of clones/reads with No mutations1.56% 13.33% 35.71% 26.17%^(b)  96.9% One mutation 98.44% 86.67% 50.0056.71%  2.75% Multiple mutations 0.00%  0.00% 14.29% 17.12% 0.034%Percent of mutated codons with 1 base substitution 14.29% 22.50% 22.72%31.97% 86.11% 2 base substitution 42.86% 47.50% 59.10% 41.84% 13.89% 3base substitution 42.86% 30.00% 18.18% 26.20%  0.00% Percent of possiblecodon substitutions observed 1 base substitution 99.96% 2 basesubstitutions 97.70% 3 base substitutions 95.33% All substitutions97.01% ^(a)two mutations were identified outside the region targeted formutagenesis ^(b)most of the reads with multiple mutations and about 50%of the reads with no mutations result from PCR jumping during ampliconcreation (see text hereinbelow).The library mostly is comprised of members with one mutation asindicated in sequencing of individual clones.

TABLE 5 Percent of bases in mutated codons in the comprehensive codonmutagenesis library CCM-1. 454 Sequencing of sequencing of Expected in90 individual 454 TEM-1 (i.e. an ideal clones of the sequencing ofsequencing Base library library library errors) G 25.00% 46.25% 43.26%26.85% A 25.00% 17.08% 18.84% 18.52% T 25.00% 18.33% 18.81% 24.54% C25.00% 18.33% 19.09% 30.09%Construction and Characterization of Comprehensive Codon MutagenesisLibrary CCM-2

To confirm that the bias for G's resulted from their overrepresentationin the mutagenic oligonucleotides, a second set of three libraries(CCM-2) was constructed using a second set of degenerate mutagenicoligonucleotides that were synthesized using a hand-mixed ratio of bases(instead of machine mixed). The three libraries were transformed intoNEB 5-alpha F′Iq cells, which contain the lacIq repressor to betterrepress expression to avoid any bias when propagating the library.Sequencing of 30 members of each library revealed 8.9% wildtype, 83.1%single codon substitutions in the targeted region, 1.1% with a singlemutation outside the targeted region, and 6.7% multiple mutations (3 of6 had two-mutations in the targeted region; 3 of 6 had one codonsubstitution in the targeted region and the second mutation in anon-targeted region). The frequency of bases substituted in the designedmutations of CCM-2 (27.5%:26.6%:23.0%:23.0% for G:A:C:T) was much moreeven than in CCM-1. The ratio of 1-base:2-base:3-base substitutions inthe targeted region was 7.7%:42.2%:30.1%.

PFunkel Error Rate

PfuTurbo Cx hotstart DNA polymerase has a published error rate of1.3×10⁻⁶ in a PCR reaction using a double-stranded template (PfuTurbo CxHotstart DNA Polymerase Instruction Manual, 2009). For site-directed andmulti-site mutagenesis using a single-stranded template, threeunintended mutations were observed outside the region of the mutagenicoligonucleotides in 77 sequencing reactions of the 861 bp TEM-1 gene,which corresponds to an error rate of 4.5×10⁻⁵. For the comprehensivecodon mutagenesis, 6 mutations were observed outside the target regionin CCM-1 and CCM-2, which corresponds to an error rate of 5.8×10⁻⁵.These error rates are 35- and 45-fold higher than PfuTurbo Cx hotstartDNA polymerase's error rate. Without wishing to be bound to any oneparticular theory, it is thought that the elevated error rate resultsfrom deviations from the recommended PfuTurbo Cx reaction buffer and/ordegradation of the ssDNA template at 95° C. All the observed unintendedmutations can be explained by cytosine deamination (approximately 2×10⁻⁷events/sec at 95° C. in ssDNA (Lindahl and Nyberg, 1972) leading toG:C->A:T transitions or depurination (approximately 4×10⁻⁷ events/sec at95° C. in ssDNA (Lindahl and Nyberg, 1972; André et al., 1997) which canlead to various mutations.

Example 5 Identification of Adaptive Codon Substitutions in TEM-1 thatConfer Increased Tazobactam Resistance with a Single Amino AcidSubstitution

An extensive knowledge of the possible molecular determinants ofbacterial resistance to β-lactam antibiotics and β-lactamase inhibitorswould inform the development and implementation of new antibiotics andinhibitors. Adaptive codon substitutions in TEM-1 were identified thatconferred increased resistance to the β-lactamase inhibitor tazobactam,which is used clinically in combination with the extended spectrumβ-lactam antibiotic piperacillin in the drug Tazocin/Zosyn. Theseadaptive mutations were identified from library CCM-2—a secondcomprehensive codon mutagenesis library that was constructed whichlacked the oligonucleotide-derived bias for G's in the substituted codonobserved in CCM-1 (FIG. 5). CCM-2 was subjected to a selection for anincrease in resistance to tazobactam. Under the selective conditions,wildtype survived at a frequency of about 10⁻³. Sequencing of 279colonies revealed 120 unique non-wildtype sequences. Since anyparticular amino acid substitution is relatively rare in the library,the criteria that an amino acid substitution had to be observed twicewas used to categorize it as potentially adaptive in nature. The set ofthese potentially adaptive substitutions (Table 6) overlapped one (M69L)but not other mutations previously found in alleles that increasetazobactam resistance, most notably R244S and N276D (Robin et al.,2011). In addition, 18 new, potentially adaptive amino acidsubstitutions, the most prevalent of which were 8 different amino acidsubstitutions at Y105 and the S235T mutation, were identified. The Y105S/D/N and S235T mutations can occur with a single base change and arethe most likely to appear naturally. These four mutations wereintroduced by single base substitution into TEM-1 and the ampicillin,piperacillin, and tazobactam resistance of these alleles were comparedto previously known tazobactam resistance alleles (FIG. 6). Data for allreplicates are shown in Tables 7 and 8. It was found that all fourprovide higher resistance to ampicillin in the presence of tazobactamthan R244S and N276D, suggesting that the selection was too strong toidentify R244S and N276D. The Y105N, Y105S, and S235T alleles showedsignificant inhibitor resistance for both ampicillin and piperacillinhydrolysis—at or above that of the M69L allele, which is the mostresistant allele observed to date for the piperacillin/tazobactamcombination (Robin et al., 2011). It is predicted that Y105N, Y105S, andS235T have the potential to emerge in the clinic. Without wishing to bebound to any one particular theory, it is believed that theirnonemergence to date, and the fact that they were not identified inprevious selections for tazobactam resistance performed on error pronePCR libraries (Vakulenko et al., 1998) may reflect the fact that therequired base substitutions are not as common as the base substitutionsfor previously identified tazobactam resistance mutations. Again,without wishing to be bound to any one particular theory, it is thoughtthat these mutations were readily identified by the presently disclosedmethods because the PFunkel mutagenesis provides a less biased and muchmore comprehensive library of mutations than error prone PCR.

TABLE 6 Potential adaptive amino acid substitutions in TEM-1 identifiedfrom genetic selections for tazobactam resistance codon substitutions.Amino acid substitutions Ambler Clinically Codon position^(a)observed^(b) This Study^(c) Occurrences^(d) coverage^(e) I13 — L 2 2 of6 L21 F.I Q 2 1 of 2 M69 L, I, V L 30 6 of 6 Q90 — A 2 1 of 4 Y105 — G14 4 of 4  S* 10 3 of 6 A 7 2 of 4 D 5 2 of 2 N 5 2 of 2 W 4 1 of 1 T 22 of 4 R120 G E 3 1 of 2 S124 N Q 2 1 of 2 T128 — E 2 1 of 2 T140 — G 21 of 4 E147 — G 2 2 of 4 W165 R, C, G Y 4 2 of 2 S235 — T 8 3 of 4 T265M M 4 1 of 1 ^(a)Numbering according to Ambler et al. (1998). ^(b)Aminoacid substitutions observed in natural alleles of TEM-1 with increasedresistance to b-lactam antibiotics of β-lactamase inhibitorshttp://www.lahey.org/studies/temtable.asp). Amino acid substitutionsunderlined are found in alleles with increased inhibitor resistance(Drawz and Bonomo, 2010). ^(c)Amino acid substitutions in bold wereobserved with a single based change in the codon. *means that althoughthe amino acid substitution can occur with a single base change, such achange was not observed here. ^(d)Of the amino acid substitution in thisstudy. ^(e)For the amino acid substitutions found in this this study,the number of unique codons observed out of the possible number ofunique codons is reported.

TABLE 7 Ampicillin MIC values for selected alleles. MIC^(a) ampicillin(μg/mL) −tazobactam +tazobactam^(b) Repli- Me- Me- cate 1 2 3 dian 1 2 3dian none 8192 8192 8192 8192 16 22.6 22.6 22.6 M69L 5792 8192 8192 8192512 724 724 724 Y105D 1448 1448 1448 1448 256 256 362 256 Y105N 40965792 5792 5792 148 1448 1448 1448 Y105S 2896 2896 2896 2896 724 10241024 1024 S235T 8192 8192 8192 8192 256 362 512 362 R244S 2896 4096 40964096 64 128 90.5 90.5 N276D 8192 8192 8192 8192 90.5 90.5 128 90.5^(a)Median value of three replicates. MIC assays performed in √2-foldincrements (Mueller Hinton broth-agar, 10⁴ CFU/spot, 37° C. for 12hours). ^(b)tazobactam added to 6 μg/mL

TABLE 8 Piperacillin MIC values for selected alleles. MIC^(a) ampicillin(μg/mL) −tazobactam +tazobactam^(b) Repli- Me- Me- cate 1 2 3 dian 1 2 3dian none 2896 2896 2896 2896 1 1.4 1.4 1.4 M69L 2048 2896 2896 289622.6 32 22.6 22.6 Y105D 1.4 1.4 1.4 1.4 1 1 1 1 Y105N 2048 2048 20482048 32 45 45 45 Y105S 1024 1448 1448 1448 16 16 16 16 S235T 2896 28962896 2896 32 16 22.6 22.6 R244S 1024 1448 1448 1448 2 2.83 2 2 N276D2896 2896 2896 2896 1 1.4 2 1.4 ^(a)Median value of three replicates.MIC assays performed in √2-fold increments (Mueller Hinton broth-agar,10⁴ CFU/spot, 37° C. for 12 hours). ^(b)tazobactam added to 6 μg/mL

Example 6 PFunkel Mutagenesis Using a dsDNA Template

In some embodiments, the methods described above require that the genetargeted for mutation is in a phagemid (a plasmid containing the flphage origin) and require the production of phage particles from whichthe dU-ssDNA template is isolated. Although preparation of such atemplate is straightforward, the presently disclosed methods can beexpanded to be applicable to any plasmid and to simplify the protocol byeliminating the need for phage entirely. The phageless PFunkelmutagenesis (FIG. 7, Table 9) achieves this by utilizing a dU-dsDNAplasmid template. After the mutation-containing second strand synthesis,UDG and ExoIII are added to degrade both strands of the dU-dsDNAtemplate. The newly-synthesized, circular ssDNA is then converted todsDNA using the reverse oligonucleotide. Like PFunkel mutagenesis usinga ssDNA template, the reaction can be performed in a single tube using athermocycler. Some of the major differences from the embodiment of thepresently disclosed methods shown in FIG. 1 are the use of a dsDNAtemplate instead of a ssDNA template and the degradation of thedU-containing template before the third strand synthesis.

PFunkel mutagenesis using a dsDNA template was tested for site-directedmutagenesis and multiple-site mutagenesis. For site-directed mutagenesis(using the c542a mutagenic primer), 708,000 transformants were obtained,and 10 of 10 randomly selected colonies had the desired mutation and noundesired mutations. For multiple-site mutagenesis, the creation of fourspecific mutations at distant sites in the gene was attempted. 445,000transformants were obtained. Four of 10 colonies had all four mutations,the remainder either were wildtype (5 colonies) or had less than 4mutations (1 colony). Without wishing to be bound to any one particulartheory, it is believed that the apparent lower efficiency of multi-sitemutagenesis using a dsDNA template may result from the difficulty ingetting all four primers to simultaneously anneal to a dsDNA template(as opposed to a ssDNA template) or difficulty in degrading the dsDNAtemplate.

TABLE 9 Reaction conditions for PFunkel mutagenesis using a dsDNAtemplate. Site-directed mutagenesis Multiple-site mutagenesis Start withPfuTurbo Cx buffer PfuTurbo Cx buffer 2.5 units PfuTurbo Cx 1 μg (0.38pmol) of dU- dsDNA polymerase 2 μL of 3 μM kinased 10 mM DTT mutagenicoligo mixture 0.5 mM NAD⁺ 77 μL total volume 0.2 mM dNTPs 1 μg (0.38pmol) of dU-dsDNA 1 μL of 1.5 μM kinased mutagenic oligo 200 cohesiveend units Taq ligase 100 μL total volume Initial step None None Cycling1 cycle of 95° C. for 3 min, 95° C. for 3 min 55° C. for 10 min 55° C.for 90 sec hold at 55° C. 68° C. for 15 min add 10 mM DTT 0.5 mMNAD^(+,) 0.2 mM dNTPs 2.5 units PfuTurbo Cx (previously heat activated)200 cohesive end units Taq ligase bringing the total volume to 100 μL65° C. for 15 min Ligation 45° C. for 15 min Degradation of Shift to 37°C. Shift to 37° C. template and side- Add 10 units of UDG + 30 units ofExoIII; add 5 units of UDG + 2 units of products incubate 1 hour at 37°C.; 70° C. for 20 min ExoIII; incubate 1 hr at 37° C.; 70° C. (heatinactivation) for 20 min (heat inactivation) add Add 3.8 pmol of kinasedoligo P320 (10:1 molar ratio oligo to template) Synthesis of 1 cycle ofsecond strand 95° C. for 30 sec containing 55° C. for 45 sec mutation68° C. for 10 min 45° C. for 15 min (ligation) Final step Purify DNA(optional step to increase number of transformants) and transform

Table 10 shows a comparison of PFunkel single-site mutagenesis with asingle-stranded (PFunkel) and double-stranded (Phage-less PFunkel)template. The results show that using either the single-stranded ordouble-stranded template resulted in a 100% mutation rate. A comparisonof multiple-site mutagenesis with a single-stranded (PFunkel) anddouble-stranded (Phage-less PFunkel) template also showed similarmutation rates (Table 11).

TABLE 10 PFunkel single mutagenesis with single-stranded (PFunkel) anddouble- stranded (Phage-less PFunkel) template Single Mutation PFunkelPhage-less PFunkel Template ssDNA dsDNA Mutagenic Oligo c542a c542aColonies with mutation 23 of 23 10 of 10 Wild-type  0 of 23  0 of 10Transformants 533,000 708,000 Template-degrading enzymes 30 units exoIII30 units exoIII

TABLE 11 PFunkel multiple site mutagenesis with single-stranded(PFunkel) and double-stranded (Phage-less PFunkel) template PFunkelMulti Template ssDNA ssDNA Mutagenic Oligos A42G A42(NNN) E104KE104(NNN) M182T M182(NNN) G238S G238(NNN) Colonies with all 3 of 5 7 of10 positions mutated Wild-type 0 of 5 0 of 10 Transformants 1,440,0005,840,000 Template-degrading 2 units exoIII 2 units exoIII enzymesPhageless PFunkel Multi Template dsDNA dsDNA dsDNA Mutagenic Oligos A42GA42(NNN) A42(NNN) E104K E104(NNN) E104(NNN) M182T M182(NNN) M182(NNN)G238S G238(NNN) G238(NNN) Colonies with all 4 of 10 2 of 13 5 of 10positions mutated Wild-type 5 of 10 8 of 13 3 of 10 Transformants445,000 140,000 160* Template- 2 units exoIII 2 units exoIII 50 unitsExoI degrading enzymes 2 units ExoIII *5 μL of the unpurified reactionwas transformed directly into chemically competent cells, therefore thenumber of transformants is lower.

Example 7 Discussion

The presently disclosed subject matter offers a very efficient methodfor site-directed mutagenesis at single or multiple-sitessimultaneously. Once the template DNA is prepared, the method can becompleted in a single day in a single tube, and requires no intermediateDNA purification or sub-cloning. PFunkel can be used for site-directedmutagenesis at an efficiency approaching 100%.

However, the real power of the PFunkel mutagenesis lies in the abilityto make extensive, user defined libraries of single or multiplemutations. For example, there currently is no efficient method to make alibrary comprising all 5700 possible single amino acid mutations of a300 amino acid long protein, nor is there a method to make auser-prescribed subset of 2000 of these 5700 mutations. The presentlydisclosed methods can be used for alanine scanning mutagenesis(Cunningham and Wells, 1989) to create all possible alaninesubstitutions, or a user-defined subset thereof in a single reaction.Expanding to scan all amino acids or all codons is equally simple.Mutating one or more specific sites simultaneously is also possible withease. Comprehensive codon mutagenesis using PFunkel efficiently makeslibraries for deep mutational scanning (Araya and Fowler, 2011) withoutthe need for the costly and time-consuming construction of separatelibraries for every codon analyzed. Compared to error prone PCR (Leunget al., 1989), which can practically access only approximately 30% ofthe possible amino acid substitutions in a gene, comprehensive codonmutagenesis allows effective access to all 100%.

The creation of a library with site-saturation at four distal sitessimultaneously at 70% efficiency has been demonstrated herein. PFunkelmutagenesis has also been employed to create a comprehensive codonmutagenesis library of the TEM-1 β-lactamase gene. This library wasdesigned to contain 18,081 members, one for each possible codonsubstitution in the gene (287 positions in TEM-1×63 possible codonsubstitutions). Deep sequencing revealed that approximately 97% of thedesigned single codon substitutions are present in the library. Fromsuch a library, 18 previously unreported adaptive mutations wereidentified, each of which confer resistance to the β-lactamase inhibitortazobactam. Three of these mutations confer resistance equal to orhigher than that of the most resistant reported TEM-1 allele and havethe potential to emerge clinically.

For directed evolution studies, the generation of diversity bycomprehensive codon mutagenesis allows access to unique mutationalpathways not readily explored by current methods—enabling theidentification of unique proteins with improved properties. PFunkelmutagenesis can efficiently create defined mutagenic diversity atmultiple sites simultaneously and is thus tailor-made for the creationof computationally designed libraries for subsequent screening orselection strategies. All amino acids and codons can be accessed at anydesired position.

PFunkel mutagenesis overcomes the limitations of currently availabletechnology to offer a convenient, highly efficient and high-throughputapproach for creating a user-defined library of gene mutants in whichsingle or multiple mutations can be located anywhere in the gene.

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Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A method for introducing one or more mutationsto a single-stranded target nucleic acid molecule, said methodcomprising: a) conducting a first amplification reaction in the presenceof a thermostable DNA polymerase and a thermostable DNA ligase at anelevated temperature to synthesize a mutagenized strand of DNAcomprising at least one mutagenic oligonucleotide relative to acomplementary single-stranded template DNA comprising a target nucleicacid molecule in a circular DNA vector; b) conducting a secondamplification reaction in the presence of a thermostable DNA polymeraseand a thermostable DNA ligase at an elevated temperature to synthesize acomplementary mutant strand of DNA; and c) degrading the template DNAand non-covalently closed circular nucleic acid molecules at an elevatedtemperature to obtain a mutation-containing double-stranded DNA product.2. The method of claim 1, further comprising annealing at least onemutagenic oligonucleotide comprising at least one mutation to the targetnucleic acid molecule at a first elevated temperature before conductingthe first amplification reaction.
 3. The method of claim 2, wherein thefirst amplification reaction is conducted in the presence of thethermostable DNA polymerase and the thermostable DNA ligase at a secondelevated temperature to synthesize the mutagenized strand of DNAcomprising the at least one mutagenic oligonucleotide.
 4. The method ofclaim 3, further comprising denaturing the mutagenized strand of DNA ata third elevated temperature before conducting the second amplificationreaction.
 5. The method of claim 4, further comprising annealing areverse primer to the mutagenized strand of DNA at a fourth elevatedtemperature after denaturing the mutagenized strand of DNA and beforeconducting the second amplification reaction.
 6. The method of claim 5,wherein the second amplification reaction is conducted in the presenceof the thermostable DNA polymerase and the thermostable DNA ligase at afifth elevated temperature to synthesize the complementary mutant strandof DNA.
 7. The method of claim 6, wherein degrading the template DNA andnon-covalently closed circular nucleic acid molecules is conducted at asixth elevated temperature to obtain the mutation-containingdouble-stranded DNA product.
 8. The method of claim 7, wherein at leastone or more of: the first elevated temperature is from about 50° C. toabout 60° C.; the second elevated temperature is from about 60° C. toabout 73° C.; the third elevated temperature is from about 90° C. toabout 98° C.; the fourth elevated temperature is from about 50° C. toabout 60° C.; the fifth elevated temperature is about 60° C. to about73° C.; and the sixth elevated temperature is from about 32° C. to about42° C.
 9. The method of claim 1, wherein degrading the template DNAoccurs prior to conducting the second amplification reaction.
 10. Themethod of claim 1, wherein the target nucleic acid molecule is denaturedat a temperature from about 90° C. to about 98° C. before annealing theat least one mutagenic oligonucleotide to the target nucleic acidmolecule.
 11. The method of claim 1, wherein the thermostable DNApolymerase is PfuTurbo Cx.
 12. The method of claim 1, wherein thethermostable DNA polymerase is selected from the group consisting of TaqDNA polymerase, Pfu DNA polymerase, PfuTurbo DNA polymerase, Tma DNApolymerase, Tli DNA polymerase, KOD DNA polymerase, JDF-3 DNApolymerase, PGB-D DNA polymerase, Tgo DNA polymerase, and Pyrolobusfurmarius DNA polymerase, and Phusion DNA polymerase.
 13. The method ofclaim 1, wherein the thermostable DNA ligase is selected from the groupconsisting of Pfu DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermusfiliformisligase, Rhodothermus marinus DNA ligase, Thermus scotoductusDNA ligase and Bacillus stearothermophilus DNA ligase.
 14. The method ofclaim 8, wherein at least one or more of the annealing the at least onemutagenic oligonucleotide step, the first amplification reaction step,the annealing the reverse primer step, and the second amplificationreaction step, are performed at the same time by thermal cycling. 15.The method of claim 8, wherein the annealing the at least one mutagenicoligonucleotide step and the first amplification reaction step areperformed more than once before the step of denaturing the mutagenizedstrand of DNA occurs, and wherein at least one additional mutagenicoligonucleotide is added to the reaction in a stepwise manner.
 16. Themethod of claim 1, wherein at least one or more of a further step afterthe first amplification reaction step and before the denaturing stepcomprises completing the ligation at a temperature from about 40° C. toabout 50° C. and a further step after the second amplification reactionstep and before the degrading step comprises completing the ligation ata temperature from about 40° C. to about 50° C.
 17. The method of claim1, wherein the mutation-containing double-stranded DNA product comprisesone mutation.
 18. The method of claim 17, wherein the efficiency ofmutagenesis is about 98% or greater and the molar ratio of mutagenicoligonucleotide to single-stranded template is from about 1:1 to about100:1.
 19. The method of claim 1, wherein the mutation-containingdouble-stranded DNA product comprises more than one mutation.
 20. Themethod of claim 1, wherein the mutation-containing double-stranded DNAproduct comprises a gene.
 21. The method of claim 20, wherein anon-comprehensive or comprehensive codon mutagenesis library is created.22. The method of claim 1, wherein the at least one mutation is asubstitution mutation, a deletion, or an addition of a DNA base.
 23. Themethod of claim 1, further comprising transforming a host cell with theDNA product from the degrading step.
 24. The method of claim 23, whereinthe DNA product is purified before being transformed into the host cell.25. The method of claim 1, wherein a DNA glycosylase, an endonuclease,and/or an exonuclease are used to degrade the template DNA andnon-covalently closed circular nucleic acid molecules.
 26. The method ofclaim 25, wherein the DNA glycosylase comprises uracil DNA glycosylase.27. The method of claim 25, wherein the endonuclease comprises arestriction endonuclease or a nicking endonuclease.
 28. The method ofclaim 25, wherein the exonuclease comprises exonuclease III.
 29. Themethod of claim 1, wherein the complementary single-stranded template isa uracil-containing complementary single-stranded template and degradingthe template DNA and non-covalently closed circular nucleic acidmolecules comprises degrading the uracil-containing DNA andnon-covalently closed circular nucleic acid molecules.
 30. A method forintroducing one or more mutations to a double-stranded target nucleicacid molecule, the method comprising: (a) conducting a firstamplification reaction in the presence of a thermostable DNA polymeraseand a thermostable DNA ligase at an elevated temperature to synthesize amutagenized strand of DNA comprising at least one mutagenicoligonucleotide relative to at least one complementary strand of adouble-stranded template comprising a target nucleic acid molecule in acircular DNA vector; (b) degrading the template DNA and non-covalentlyclosed circular nucleic acid molecules at an elevated temperature; and(c) conducting a second amplification reaction in the presence of athermostable DNA polymerase and a thermostable DNA ligase at an elevatedtemperature to synthesize a complementary mutant strand of DNA andobtain a mutation-containing double-stranded DNA product.
 31. The methodof claim 30, further comprising denaturing the target nucleic acidmolecule at a first elevated temperature before conducting the firstamplification reaction.
 32. The method of claim 31, further comprisingannealing the at least one mutagenic oligonucleotide comprising the atleast one mutation to the target nucleic acid molecule at a secondelevated temperature after denaturing the target nucleic acid moleculeand before conducting the first amplification reaction.
 33. The methodof claim 32, wherein the first amplification reaction is conducted inthe presence of the thermostable DNA polymerase and the thermostable DNAligase at a third elevated temperature to synthesize the mutagenizedstrand of DNA comprising the at least one mutagenic oligonucleotide. 34.The method of claim 33, further comprising denaturing the mutagenizedstrand of DNA at a fifth elevated temperature after degrading thetemplate DNA and non-covalently closed circular nucleic acid molecules.35. The method of claim 34, further comprising annealing a reverseprimer to the denatured mutagenized strand of DNA at a sixth elevatedtemperature before conducting the second amplification reaction.
 36. Themethod of claim 35, wherein the second amplification reaction isconducted in the presence of the thermostable DNA polymerase and thethermostable DNA ligase at a seventh elevated temperature to synthesizethe complementary mutant strand of DNA and obtain themutation-containing double-stranded DNA product.
 37. The method of claim36, wherein at least one or more of: the first elevated temperature isfrom about 90° C. to about 98° C.; the second elevated temperature isfrom about 50° C. to about 60° C.; the third elevated temperature isfrom about 60° C. to about 73° C.; the fourth elevated temperature isfrom about 32° C. to about 42° C.; the fifth elevated temperature isfrom about 90° C. to about 98° C.; the sixth elevated temperature isfrom about 50° C. to about 60° C.; and the seventh elevated temperatureis from about 60° C. to about 73° C.
 38. The method of claim 30, whereinthe thermostable DNA polymerase is PfuTurbo Cx.
 39. The method of claim30, wherein the thermostable DNA polymerase is selected from the groupconsisting of Taq DNA polymerase, Pfu DNA polymerase, PfuTurbo DNApolymerase, Tma DNA polymerase, Tli DNA polymerase, KOD DNA polymerase,JDF-3 DNA polymerase, PGB-D DNA polymerase, Tgo DNA polymerase, andPyrolobus furmarius DNA polymerase, and Phusion DNA polymerase.
 40. Themethod of claim 30, wherein the thermostable DNA ligase is selected fromthe group consisting of: Pfu DNA ligase, Tth DNA ligase, Taq DNA ligase,Thermus filiformisligase, Rhodothermus marinus DNA ligase, Thermusscotoductus DNA ligase and Bacillus stearothermophilus DNA ligase. 41.The method of claim 30, wherein at least one or more of the annealingthe at least one mutagenic oligonucleotide step, the first amplificationreaction step, the annealing the reverse primer step, and the secondamplification reaction step are performed at the same time.
 42. Themethod of claim 30, wherein the annealing the at least one mutagenicoligonucleotide step and the first amplification reaction step areperformed more than once before the degrading step occurs, and whereinat least one additional mutagenic oligonucleotide is added to thereaction in a stepwise manner.
 43. The method of claim 30, wherein atleast one or more of a further step after the first amplificationreaction step and before the degrading step and a further step after thesecond amplification reaction step comprises completing the ligation ata temperature from about 40° C. to about 50° C.
 44. The method of claim30, wherein the mutation-containing double-stranded DNA productcomprises one mutation and the efficiency of mutagenesis is about 98% orgreater.
 45. The method of claim 44, wherein the molar ratio ofmutagenic oligonucleotide to double-stranded template is from about 1:1to about 100:1.
 46. The method of claim 30, wherein themutation-containing double-stranded DNA product comprises more than onemutation.
 47. The method of claim 30, wherein the mutation-containingdouble-stranded DNA product comprises a gene.
 48. The method of claim47, wherein a non-comprehensive or comprehensive codon mutagenesislibrary is created.
 49. The method of claim 48, wherein thenon-comprehensive or comprehensive codon mutagenesis library is used toobtain an improved protein, gene or gene promoter.
 50. The method ofclaim 30, wherein the at least one mutation is a substitution mutation,a deletion, or an addition of a DNA base.
 51. The method of claim 30,wherein the complementary double-stranded template is auracil-containing complementary double-stranded template and degradingthe template DNA and non-covalently closed circular nucleic acidmolecules comprises degrading the uracil-containing DNA andnon-covalently closed circular nucleic acid molecules.
 52. The method ofclaim 30, wherein a DNA glycosylase, an endonuclease, and/or anexonuclease are used to degrade the template DNA and non-covalentlyclosed circular nucleic acid molecules.
 53. The method of claim 52,wherein the DNA glycosylase comprises uracil DNA glycosylase.
 54. Themethod of claim 52, wherein the endonuclease comprises a restrictionendonuclease or a nicking endonuclease.
 55. The method of claim 52,wherein the exonuclease comprises exonuclease III.